Methods for screening compounds that affect IL-1 epsilon activity

ABSTRACT

The invention is directed to purified and isolated novel human IL-1 epsilon polypeptides, the nucleic acids encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, the use of such polypeptides in cellular and immune reactions, the use of such polypeptides in screening for agonists or antagonists of IL-1 epsilon activity, and kits comprising such polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. applications Ser.Nos. 60/097,413, 60/098,595, 60/099,974, 09/763,498, (PCT/US99/18771)and No. 60/313,110 filed Aug. 21, 1998, Aug. 31, 1998, Sep. 11, 1998,May 15, 2001, (Aug. 20, 1999) and Aug. 16, 2001, respectively. Theentire disclosures of these applications are relied upon andincorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is directed to purified and isolated novel human IL-1epsilon polypeptides, the nucleic acids encoding such polypeptides,processes for production of recombinant forms of such polypeptides,antibodies generated against these polypeptides, the use of suchpolypeptides in cellular and immune reactions, the use of suchpolypeptides in screening for agonists or antagonists of IL-1 epsilonactivity, and kits comprising such polypeptides.

2. Description of Related Art

Interleukin-1 (IL-1) is a member of a large group of cytokines whoseprimary function is to mediate immune and inflammatory responses. Thereare five known IL-1 family members which include IL-1 alpha, IL-1 beta,IL-1 receptor antagonist (IL-1ra), IL-1 delta (as disclosed in PCTUS/99/00514), and IL-18 (previously known as IGIF and sometimes IL-1gamma). IL-1 that is secreted by macrophages is actually a mixture ofmostly IL-1 beta and some IL-1 alpha (Abbas et al., 1994). IL-1 alphaand IL-1 beta, which are first produced as 33 kD precursors that lack asignal sequence, are further processed by proteolytic cleavage toproduce secreted active forms, each about 17 kD. Additionally, the 33 kDprecursor of IL-1 alpha is also active. Both forms of IL-1 are theproducts of two different genes located on chromosome 2. Although thetwo forms are less than 30 percent homologous to each other, they bothbind to the same receptors and have similar activities.

IL-1ra, a biologically inactive form of IL-1, is structurally homologousto IL-1 and binds to the same receptors. Additionally, IL-1ra isproduced with a signal sequence which allows for efficient secretioninto the extracellular region where it competitively competes with IL-1(Abbas et al., 1994).

The IL-1 family ligands bind to two IL-1 receptors that are members ofthe Ig superfamily. IL-1 receptors include the 80 kDa type II receptor(IL-1RII) and a 68 kDa type II receptor (IL-1RII). The ligands also bindto a soluble proteolytic fragment of IL-1RII (sIL-1RII) (Colotta et al.,Science 261(5120):472-75, 1993).

The major source of IL-1 is the activated macrophage or mononuclearphagocyte. Other cells that produce IL-1 include epithelial andendothelial cells (Abbas et al., 1994). IL-1 secretion from macrophagesoccurs after the macrophage encounters and ingests gram-negativebacteria. Such bacteria contain lipopolysaccharide (LPS) molecules, alsoknown as endotoxin, in the bacterial cell wall. LPS molecules are theactive components that stimulate macrophages to produce tumor necrosisfactor (TNF) and IL-1. In this case, IL-1 is produced in response to LPSand TNF production. At low concentrations, LPS stimulates macrophagesand activates B-cells and other host responses needed to eliminate thebacterial infection; however, at high concentrations, LPS can causesevere tissue damage, shock, and even death.

The biological functions of IL-1 include activating vascular endothelialcells and lymphocytes, local tissue destruction, and fever (Janeway etal., 1996). At low levels, IL-1 stimulates macrophages and vascularendothelial cells to produce IL-6, upregulates molecules on the surfaceof vascular endothelial cells to increase leukocyte adhesion, andindirectly activates inflammatory leukocytes by stimulating mononuclearphagocytes and other cells to produce certain chemokines that activateinflammatory leukocytes. These IL-1 functions are crucial during lowlevel microbial infections. However, if the microbial infectionescalates, IL-1 acts systemically by inducing fever, stimulatingmononuclear phagocytes to produce IL-1 and IL-6, increasing theproduction of serum proteins from hepatocytes, and activating thecoagulation system. It is also known that IL-1 does not causehemorrhagic necrosis of tumors or suppress bone marrow stem celldivision. Nevertheless, IL-1 is lethal to humans at high concentrations.

Given the important function of IL-1, there is a need in the art foradditional members of the IL-1 ligand family. In addition, in view ofthe continuing interest in protein research and the immune system, thediscovery, identification, and roles of new proteins, such as human IL-1epsilon and its receptors, are at the forefront of modern molecularbiology and biochemistry. Despite the growing body of knowledge, thereis still a need in the art for the identity and function of proteinsinvolved in cellular and immune responses.

SUMMARY OF THE INVENTION

The invention aids in fulfilling these needs in the art by providingisolated human IL-1 epsilon nucleic acids and polypeptides encoded bythese nucleic acids. Specifically, the invention encompasses an isolatedhuman IL-1 epsilon nucleic acid molecule comprising the DNA sequences ofSEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:12 and an isolated human IL-1epsilon nucleic acid molecule encoding the amino acid sequence of SEQ IDNO:6, SEQ ID NO:8, and SEQ ID NO:13, as well as nucleic acid moleculescomplementary to these sequences. Both single-stranded anddouble-stranded RNA and DNA nucleic acid molecules are encompassed bythe invention, as well as nucleic acid molecules that hybridize to adenatured, double-stranded DNA relating to SEQ ID NO:5, SEQ ID NO:7, orSEQ ID NO:12. Also encompassed are isolated nucleic acid molecules thatare derived by in vitro mutagenesis from SEQ ID NO:5, SEQ ID NO:7, orSEQ ID NO:12, are degenerate from SEQ ID NO:5, SEQ ID NO:7, or SEQ IDNO:12, are allelic variants of human DNA of the invention, and arespecies homologs of DNA of the invention. The invention also encompassesrecombinant vectors that direct the expression of these nucleic acidmolecules and host cells transformed or transfected with these vectors.In addition, the invention encompasses methods of using the nucleic acidnoted above in assays to identify chromosomes, map human genes, andstudy the immune system.

The invention also encompasses isolated polypeptides encoded by thesenucleic acid molecules, synthetic polypeptides encoded by these nucleicacid molecules, and peptides and fragments of these polypeptides.Isolated polyclonal or monoclonal antibodies that bind to thesepolypeptides are also encompassed by the invention. The inventionfurther encompasses methods for the production of IL-1 epsilonpolypeptides, including culturing a host cell under conditions promotingexpression and recovering the polypeptide from the culture medium.Especially, the expression of IL-1 epsilon polypeptides in bacteria,yeast, plant, insect, and animal cells is encompassed by the invention.

In general, the polypeptides of the invention can be used to studycellular processes such as immune regulation, cell proliferation, celldeath, and inflammatory responses. In addition, the IL-1 epsilon ligandpolypeptides of the invention (including fragments of IL-1 epsilon), canbe used to identify proteins associated with IL-1-like ligands andIL-1-like receptors.

In addition, assays utilizing IL-1 epsilon ligand polypeptides of theinvention (including fragments of IL-1 epsilon) to screen for potentialinhibitors and/or agonists of activity associated with polypeptidecounter-structure molecules, and methods of using the inventive IL-1epsilon ligand polypeptides as therapeutic agents for the treatment ofdiseases mediated by IL-1 epsilon ligand polypeptide counter-structuremolecules are encompassed by the invention. Further, methods of usingIL-1 epsilon ligand polypeptides of the invention in the design ofinhibitors and/or agonists thereof are also an aspect of the invention.

Further encompassed by this invention are kits to aid in thesedeterminations.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will be more fully described with reference to thedrawings in which:

FIG. 1 is the nucleotide sequences of human IL-1 epsilon DNA of theinvention, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:12.

FIG. 2 is the amino acid sequence of polypeptides, SEQ ID NO:6, SEQ IDNO:8, and SEQ ID NO:13, encoded by the nucleotide sequences of SEQ IDNO:5, SEQ ID NO:7, and SEQ ID NO:12, respectively.

FIG. 3 depicts the amino acid homology between the 3′ exon of human IL-1epsilon and murine IL-1 epsilon (long form).

FIG. 4 depicts the amino acid homology between human IL-1 epsilon (aminoacids 51-159) and murine IL-1 epsilon (long form).

DETAILED DESCRIPTION OF THE INVENTION

Interleukin-1 (IL-1) receptors are members of the large Ig superfamilyof cytokine receptors, many of which mediate the response of immunesystem cells, in particular lymphocytes. In recent years, members of thefamily of ligands that bind to these receptors have been discovered atan accelerated pace. The increase in the number of known IL-1 ligandshas been largely due to the advent of gene cloning and sequencingtechniques. Amino acid sequences deduced from nucleotide sequences areconsidered to represent IL-1 ligands if they share homology with otherknown IL-1 ligands.

Mouse IL-1 epsilon is a homolog of the known IL-1 genes, IL-1 alpha,IL-1 beta, IL-1delta (disclosed in PCT US/99/00514) and IL-1ra, and morerecently, IL-18, previously known sometimes as IL-1 gamma. Mouse IL-1epsilon was first identified by searching the EST database, anddiscovering an EST corresponding to mouse IL-1 epsilon (accession numberAA030324). The entire open reading frame for the “long form” (see below)is contained in this EST.

Mouse IL-1 epsilon (Long Form) DNA Sequence ATGTTCAGCA TCTTAGTAGTCGTGTGTGGA TCCTGCAGAA CAATATCCTC (SEQ ID NO: 1) ACTGCAGTCC CAAGGAAAGAGCAAACAGTT CCAGGAAGGG AACATAATGG AAATGTACAA CAAAAAGGAA CCTGTAAAAGCCTCTCTCTT CTATCACAAG AAGAGTGGTA CAACCTCTAC ATTTGAGTCT GCAGCCTTCCCTGGTTGGTT CATCGCTGTC TGCTCTAAAG GGAGCTGCCC ACTCATTCTG ACCCAAGAACTGGGGGAAAT CTTCATCACT GACTTCGAGA TGATTGTCGT ACATTAA Mouse IL-1 epsilon(Long Form) Amino Acid Sequence MFRILVVVCG SCRTISSLQS QGKSKQFQEGNIMEMYNKKE PVKASLFYHK (SEQ ID NO: 2) KSGTTSTFES AAFPGWFIAV CSKGSCPLILTQELGEIFIT DFEMIVVH*

While showing homology to the IL-1 genes, mouse IL-1 epsilon is unusualin that the EST originally identified appeared to encode the C-terminaltwo-thirds of an IL-1-like molecule. In addition, during studies of theexpression of IL-1 epsilon, it became apparent that there are two,alternatively spliced, forms of mRNA that encode proteins with identicalN-termini but divergent C-termini. The longer of these two proteins wasthat encoded by the original EST. The shorter (sometimes called the“isoform”) is approximately one-third the length of a typical IL-1family molecule.

Mouse IL-1 epsilon (Short Form) DNA Sequence ATGTTCAGGA TCTTAGTAGTCGTGTGTGGA TCCTGCAGAA CAATATCCTC (SEQ ID NO: 3) ACTGCAGTCC CAAGGAAAGAGCAAACAGTT CCAGTCACTA TTACCTTGCT CCCATGCCAA TATCTGGACA CTCTTGAGACGAACAGGGGG GATCCCACGT ACATGGGAGT GCAAAGGCCG ATGA Mouse IL-1 epsilon(Short Form) Amino Acid Sequence MFRILVVVCG SCRTISSLQS QGKSKQFQSLLPCSHANIWT LLRRTGGIPR (SEQ ID NO: 4) TWECKGR*

These two proteins (the long and the short form), encoded byalternatively spliced versions of the same original RNA transcript, mayassociate non-covalently and thus form a “whole” IL-1-like molecule.

In any event, using as a probe the mixed cDNAs for mouse long-form andshort-form IL-1 epsilon, human IL-1 epsilon has been identified byscreening of a human genomic library. Sequencing of a clone obtainedfrom the human genomic library reveals a stretch of DNA which containsan open reading frame, encoding a portion of a protein with highhomology to mouse IL-1 epsilon in the same region. The open readingframe appears to be an exon (the 3′ most exon of the coding region). Thesplice acceptor site at the 5′ end of this exon is in the identicalposition to the splice acceptor site of the corresponding exon in mouseIL-1 epsilon.

The DNA and amino acid sequences of this exon corresponding to humanIL-1 epsilon are set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively.

Nucleotide Sequence of Human IL-1 epsilon DNA: GAAAAGGATA TAATGGATTTGTACAACCAA CCCGAGCCTG TGAAGTCCTT (SEQ ID NO: 5) TCTCTTCTAC CACAGCCAGAGTGGCAGGAA CTCCACCTTC GAGTCTGTCG CTTTCCCTGG CTGGTTCATC GCTGTCAGCTCTGAAGGACG CTGTCCTCTC ATCCTTACCC AAGAACTGGG GAAAGCCAAC ACTACTGACTTTGGGTTAAC TATGCTGTTT TAA

A preferred polypeptide encoded by the nucleic acid sequence is setforth below:

Amino Acid Sequence of Human IL-1 epsilon: Translation in relevantreading frame (5′ 3′0: EKDIMDLYNQ PEPVKSFLFY HSQSGRNSTF ESVAFPGWFIAVSSEGGCPL (SEQ ID NO: 6) ILTQELGKAN TTDFGLTMLF *

The full-length human IL-1 epsilon DNA sequence was isolated asdescribed in Example I. The DNA and amino acid sequence of thefull-length human IL-1 epsilon are set forth in SEQ ID NO:7 and SEQ IDNO:8, respectively.

Full-Length Nucleotide sequence of Human IL-1 epsilon DNA: ATGGAAAAAGCATTGAAAAT TGACACACCT CAGCAGGGGA GCATTCAGGA (SEQ ID NO: 7) TATCAATCATCGGGTGTGGG TTCTTCAGGA CCAGACGCTC ATAGCAGTCC CGAGGAAGGA CCGTATGTCTCCAGTCACTA TTGCCTTAAT CTCATGCCGA CATGTGGAGA CCCTTGAGAA AGACAGAGGGAACCCCATCT ACCTGGGCCT GAATGGACTC AATCTCTGCC TGATGTGTGC TAAAGTCGGGGACCAGCCCA CACTGCAGCT GAAGGAAAAG GATATAATGG ATTTGTACAA CCAACCCGAGCCTGTGAAGT CCTTTCTCTT CTACCACAGC CAGAGTGGCA GGAACTCCAC CTTCGAGTCTGTGGCTTTCC CTGGCTGGTT CATCGCTGTC AGCTCTGAAG GAGGCTGTCC TCTCATCCTTACCCAAGAAC TGGGGAAAGC CAACACTACT GACTTTGGGT TAACTATGCT GTTTTAAFull-Length Amino Acid Sequence of Human IL-1 epsilon: Translation inrelevant reading frame (5′ to 3′): MEKALKIDTP QQGSIQDINH RVWVLQDQTLIAVPRKDRMS PVTIALISCR (SEQ ID NO: 8) HVETLEKDRG NPIYLGLNGL NLCLMCAKVGDQPTLQLKEK DIMDLYNQPE PVKSFLFYHS QSGRNSTFES VAFPGWFIAV SSEGGCPLILTQELGKANTT DFGLTMLF*

In addition, a single nucleotide polymorphism was identified in thehuman IL-1 epsilon gene. Specifically, the polymorphism comprises anadenosine to guanosine substitution at nucleotide 35. The polypeptideencoded by this polymorphic IL-1 epsilon gene has an arginine residue atposition 12 rather than a glutamine residue. The DNA sequence of thehuman IL-1 epsilon gene containing this single nucleotide polymorphismis set forth in SEQ ID NO:12, and the full-length amino acid sequencecorresponding to this polymorphic gene is set forth in SEQ ID NO:13.

Ful1-Length Nucleotide sequence of Polymorphic Human IL-1 epsilon DNA:ATGGAAAAAG CATTGAAAAT TGACACACCT CAGCGGGGGA GCATTCAGGA (SEQ ID NO: 12)TATCAATCAT CGGGTGTGGG TTCTTCAGGA CCAGACGCTC ATAGCAGTCC CGAGGAAGGACCGTATGTCT CCAGTCACTA TTGCCTTAAT CTCATGCCGA CATGTGGAGA CCCTTGAGAAAGACAGAGGG AACCCCATCT ACCTGCGCCT GAATGGACTC AATCTCTGCC TGATGTGTGCTAAAGTCGGG GACCAGCCCA CACTGCAGCT GAAGGAAAAG GATATAATGG ATTTGTACAACCAACCCGAG CCTGTGAAGT CCTTTCTCTT CTACCACAGC CAGAGTCGCA GGAACTCCACCTTCGAGTCT GTGGCTTTCC CTGGCTGGTT CATCGCTGTC AGCTCTGAAG GAGGCTGTCCTCTCATCCTT ACCCAAGAAC TGGGGAAAGC CAACACTACT GACTTTGGGT TAACTATGCTGTTTTAA Full-Length Amino Acid Sequence of Human IL-1 epsilon:Translation in relevant reading frame (5′ to 3′): MEKALKIDTP QRGSIQDINHRVWVLQDQTL IAVPRKDRMS PVTIALISCR (SEQ ID NO: 13) HVETLEKDRG NPIYLGLNGLNLCLMCAKVG DQPTLQLKEK DIMDLYNQPE PVKSFLFYHS QSGRNSTFES VAFPGWFIAVSSEGGCPLIL TQELGKANTT DFGLTMLF*

The discovery of this DNA encoding IL-1 epsilon enables the constructionof expression vectors comprising nucleic acid sequences encoding IL-1epsilon polypeptides of the invention; host cells transfected ortransformed with the expression vectors; biologically active human IL-1epsilon polypeptides and molecular weight markers as isolated andpurified proteins; and antibodies immunoreactive with polypeptides ofthe invention.

Nucleic Acid Molecules

In a particular embodiment, the invention relates to certain isolatednucleotide sequences. A “nucleotide sequence” refers to a polynucleotidemolecule in the form of a separate fragment or as a component of alarger nucleic acid construct, that has been derived from DNA or RNAisolated at least once in substantially pure form (i.e., free ofcontaminating endogenous materials) and in a quantity or concentrationenabling identification, manipulation, and recovery of its componentnucleotide sequences by standard biochemical methods (such as thoseoutlined in Sambrook et al., Molecular Cloning: A Laboratory Manual,2^(nd) ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1989)). Such sequences are preferably provided and/or constructed inthe form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, that are typically present ineukaryotic genes. Sequences of non-translated DNA can be present 5′ or3′ from an open reading frame, where the same do not interfere withmanipulation or expression of the coding region.

Particularly preferred nucleotide sequences of the invention are SEQ IDNO:5, SEQ ID NO:7, and SEQ ID NO:12, as set forth above. The inventionfurther encompasses isolated fragments and oligonucleotides derived fromthe nucleotide sequences of SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:12.Nucleic acid sequences within the scope of the invention includeisolated DNA and RNA sequences that hybridize to the native nucleotidesequences disclosed herein under conditions of moderate or severestringency, and which encode polypeptides or fragments thereof of theinvention. These isolated DNA and RNA sequences also include full lengthDNA or RNA molecules encoding for IL-1 epsilon polypeptides.

As used herein, conditions of moderate stringency, as known to thosehaving ordinary skill in the art, and as defined by Sambrook et al.Molecular Cloning: A Laboratory Manual, 2^(nd) ed. Vol. 1, pp.1.101-104, Cold Spring Harbor Laboratory Press, (1989), include use of aprewashing solution for the nitrocellulose filters 5× SSC, 0.5% SDS, 1.0mM EDTA (pH 8.0), hybridization conditions of 50% formamide, 6× SSC at42□C (or other similar hybridization solution, such as Stark's solution,in 50% formamide at 42□C), and washing conditions of about 60□C, 0.5×SSC, 0.1% SDS. Conditions of high stringency are defined ashybridization conditions as above, and with washing at 68□C, 0.2× SSC,0.1% SDS. The skilled artisan will recognize that the temperature andwash solution salt concentration can be adjusted as necessary accordingto factors such as the length of the probe.

Due to the known degeneracy of the genetic code, wherein more than onecodon can encode the same amino acid, a DNA sequence can vary from thatshown in SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:12, and still encode apolypeptide having the amino acid sequence of SEQ ID NO:6, SEQ ID NO:8,or SEQ ID NO:13, respectively. Such variant DNA sequences can resultfrom silent mutations (e.g., occurring during PCR amplification) or canbe the product of deliberate mutagenesis of a native sequence.

The invention thus provides equivalent isolated DNA sequences encodingpolypeptides of the invention, selected from: (a) DNA derived from thecoding region of a native mammalian gene; (b) cDNA comprising thenucleotide sequence of SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:12; (c)DNA encoding the polypeptides of SEQ ID NO:6, SEQ ID NO:8, or SEQ IDNO:13; (d) DNA capable of hybridization to a DNA of (a), (b), or (c)under conditions of moderate stringency and which encodes polypeptidesof the invention; and (e) DNA which is degenerate as a result of thegenetic code to a DNA defined in (a), (b), (c), or (d) and which encodespolypeptides of the invention. Of course, polypeptides encoded by suchequivalent DNA sequences are encompassed by the invention.

DNA that is equivalent to the DNA sequence of SEQ ID NO:5, SEQ ID NO:7,or SEQ ID NO:12 will hybridize under moderately stringent conditions tothe double-stranded native DNA sequence that encode polypeptidescomprising amino acid sequences of SEQ ID NO:6, SEQ ID NO:8, or SEQ IDNO:13. Examples of polypeptides encoded by such DNA, include, but arenot limited to, polypeptide fragments and polypeptides comprisinginactivated N-glycosylation site(s), inactivated protease processingsite(s), or conservative amino acid substitution(s), as described below.Polypeptides encoded by DNA derived from other mammalian species,wherein the DNA will hybridize to the complement of the DNA of SEQ IDNO:5, SEQ ID NO:7, or SEQ ID NO:12, are also encompassed.

Expression

The nucleic acid sequence encoding polypeptides of the invention can beinserted into recombinant expression vectors using well known methods.The expression vectors include a DNA sequence of the invention operablylinked to suitable transcriptional or translational regulatorynucleotide sequences, such as those derived from a mammalian, microbial,viral, or insect gene. Examples of regulatory sequences includetranscriptional promoters, operators, or enhancers, an mRNA ribosomalbinding site, and appropriate sequences which control transcription andtranslation initiation and termination. Nucleotide sequences are“operably linked” when the regulatory sequence functionally relates tothe DNA sequence of the invention. Thus, a promoter nucleotide sequenceis operably linked to a DNA sequence if the promoter nucleotide sequencecontrols the transcription of the DNA sequence of the invention. Theability to replicate in the desired host cells, usually conferred by anorigin of replication, and a selection gene by which transformants areidentified can additionally be incorporated into the expression vector.

In addition, sequences encoding appropriate signal peptides that are notnaturally associated with polypeptides of the invention can beincorporated into expression vectors. For example, a DNA sequence for asignal peptide (secretory leader) can be fused in-frame to thenucleotide sequence of the invention so that the polypeptide isinitially translated as a fusion protein comprising the signal peptide.A signal peptide that is functional in the intended host cells enhancesextracellular secretion of the polypeptide. The signal peptide can becleaved from the polypeptide upon secretion of polypeptide from thecell.

Suitable host cells for expression of polypeptides of the inventioninclude prokaryotes, yeast or higher eukaryotic cells. Appropriatecloning and expression vectors for use with bacterial, fungal, yeast,and mammalian cellular hosts are described, for example, in Pouwels etal. Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., (1985).Cell-free translation systems could also be employed to producepolypeptides of the invention using RNAs derived from DNA constructsdisclosed herein.

Prokaryotic Systems

Prokaryotes include gram negative or gram positive organisms. Suitableprokaryotic host cells for transformation include, for example,Escherichia coli, Bacillus subtilis, Salmonella typhimurium, and variousother species within the genera Bacillus, Pseudomonas, Streptomyces, andStaphylococcus. In a prokaryotic host cell, such as Escherichia coli, apolypeptide of the invention can include an N-terminal methionineresidue to facilitate expression of the recombinant polypeptide in theprokaryotic host cell. The N-terminal Met can be cleaved from theexpressed recombinant polypeptide.

Expression vectors for use in prokaryotic host cells also generallycomprise one or more phenotypic selectable marker genes. A phenotypicselectable marker gene is, for example, a gene encoding a protein thatconfers antibiotic resistance or that supplies an autotrophicrequirement. Examples of useful expression vectors for prokaryotic hostcells include those derived from commercially available plasmids such asthe cloning vector pBR322 (ATCC 37017). pBR322 contains genes forampicillin and tetracycline resistance and thus provides simple meansfor identifying transformed cells. To construct an expression vectorusing pBR322, an appropriate promoter and a DNA sequence of theinvention are inserted into the pBR322 vector. Other commerciallyavailable vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis.,USA). Other commercially available vectors include those that arespecifically designed for the expression of proteins; these wouldinclude pMAL-p2 and pMAL-c2 vectors that are used for the expression ofproteins fused to maltose binding protein (New England Biolabs, Beverly,Mass., USA).

The promoter sequences commonly used for recombinant prokaryotic hostcell expression vectors include beta-lactamase (penicillinase), lactosepromoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al.,Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al.,Nucl. Acids Res. 8:4057, 1980; and EP-A-36776), and tac promoter(Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, p. 412, 1982). A particularly useful prokaryotic host cellexpression system employs a phage lambda P_(L) promoter and a cI857tsthermolabile repressor sequence. Plasmid vectors available from theAmerican Type Culture Collection, which incorporate derivatives of thelambda PL promoter, include plasmid pHUB2 (resident in E. coli strainJMB9 (ATCC 37092)) and pPLc28 (resident in E. coli RR1 (ATCC 53082)).

The DNA of the invention can be cloned in-frame into the multiplecloning site of an ordinary bacterial expression vector. Ideally thevector contains an inducible promoter upstream of the cloning site, suchthat addition of an inducer leads to high-level production of therecombinant protein at a time of the investigator's choosing. For someproteins, expression levels can be boosted by incorporation of codonsencoding a fusion partner (such as hexahistidine) between the promoterand the gene of interest.

For expression of the recombinant protein, the bacterial cells arepropagated in growth medium until reaching a pre-determined opticaldensity. Expression of the recombinant protein is then induced, e.g., byaddition of IPTG (isopropyl-b-D-thiogalactopyranoside), which activatesexpression of proteins from plasmids containing a lac operator/promoter.After induction (typically for 1-4 hours), the cells are harvested bypelleting in a centrifuge, e.g. at 5,000×G for 20 minutes at 4 degreesC.

For recovery of the expressed protein, the pelleted cells may beresuspended in ten volumes of 50 mM Tris-HCl (pH 8)/1 M NaCl and thenpassed two or three times through a French press. Most highly-expressedrecombinant proteins form insoluble aggregates known as inclusionbodies. Inclusion bodies can be purified away from the soluble proteinsby pelleting in a centrifuge at 5,000×G for 20 minutes, 4 degrees C. Theinclusion body pellet is washed with 50 mM Tris-HCl (pH 8)/1% TritonX-100 and then dissolved in 50 mM Tris-HCl (pH 8)/8 M urea/0.1 M DTT.Any material that cannot be dissolved is removed by centrifugation(10,000×G for 20 minutes, 20□C). The protein of interest will, in mostcases, be the most abundant protein in the resulting clarifiedsupernatant. This protein may be “refolded” into the active conformationby dialysis against 50 mM Tris-HCl (pH 8)/5 mM CaCl₂/5 MM Zn(OAc)_(γ)/1mM GSSG/0.1 mM GSH. After refolding, purification can be carried out bya variety of chromatographic methods such as ion exchange or gelfiltration. In some protocols, initial purification may be carried outbefore refolding. As an example, hexahistidine-tagged fusion proteinsmay be partially purified on immobilized nickel.

While the preceding purification and refolding procedure assumes thatthe protein is best recovered from inclusion bodies, those skilled inthe art of protein purification will appreciate that many recombinantproteins are best purified out of the soluble fraction of cell lysates.In these cases, refolding is often not required, and purification bystandard chromatographic methods can be carried out directly.

Yeast Systems

Polypeptides of the invention alternatively can be expressed in yeasthost cells, preferably from the Saccharomyces genus (e.g., S.cerevisiae). Other genera of yeast, such as Pichia, K. lactis, orKluyveromyces, can also be employed. Yeast vectors will often contain anorigin of replication sequence from a 2 μ yeast plasmid, an autonomouslyreplicating sequence (ARS), a promoter region, sequences forpolyadenylation, sequences for transcription termination, and aselectable marker gene. Suitable promoter sequences for yeast vectorsinclude, among others, promoters for metallothionein, 3-phosphoglyceratekinase (Hitzeman et al., J. Biol. Chem. 255:2073, 1980), or otherglycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; andHolland et al., Biochem. 17:4900, 1978), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other suitable vectors andpromoters for use in yeast expression are further described in Hitzeman,EPA-73,657 or in Fleer et. al., Gene, 107:285-195 (1991); and van denBerg et. al., Bio/Technology, 8:135-139 (1990). Another alternative isthe glucose-repressible ADH2 promoter described by Russell et al. (J.Biol. Chem. 258:2674, 1982) and Beier et al. (Nature 300:724, 1982).Shuttle vectors replicable in both yeast and E. coli can be constructedby inserting DNA sequences from pBR322 for selection and replication inE. coli (Amp^(r) gene and origin of replication) into theabove-described yeast vectors.

The yeast alpha-factor leader sequence can be employed to directsecretion of a polypeptide of the invention. The alpha-factor leadersequence is often inserted between the promoter sequence and thestructural gene sequence. See, e.g., Kurjan et al., Cell 30:933, 1982;Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, 1984; U.S. Pat. No.4,546,082; and EP 324,274. Other leader sequences suitable forfacilitating secretion of recombinant polypeptides from yeast hosts areknown to those of skill in the art. A leader sequence can be modifiednear its 3′ end to contain one or more restriction sites. This willfacilitate fusion of the leader sequence to the structural gene.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci.USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 μg/ml adenine, and 20 μg/ml uracil.

Yeast host cells transformed by vectors containing ADH2 promotersequence can be grown for inducing expression in a “rich” medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the ADH2 promoter occurs when glucose isexhausted from the medium.

Mammalian and Insect Systems

Alternatively, mammalian or insect host cell culture systems can beemployed to express recombinant polypeptides of the invention.Baculovirus systems for production of heterologous proteins in insectcells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).Established cell lines of mammalian origin also can be employed.Examples of suitable mammalian host cell lines include the COS-7 line ofmonkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981),L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary(CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and theCV-1/EBNA-1 cell line (ATCC CRL 10478) derived from the African greenmonkey kidney cell line CVI (ATCC CCL 70) as described by McMahan et al.(EMBO J. 10: 2821, 1991).

Established methods for introducing DNA into mammalian cells have beendescribed (Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990, pp.15-69). Additional protocols using commercially available reagents, suchas Lipofectamine (Gibco/BRL) or Lipofectamine-Plus, can be used totransfect cells (Felgner et al., Proc. Natl. Acad. Sci. USA84:7413-7417, 1987). In addition, electroporation can be used totransfect mammalian cells using conventional procedures, such as thosein Sambrook et al. Molecular Cloning: A Laboratory Manual, 2^(nd) ed.Vol. 1-3, Cold Spring Harbor Laboratory Press, 1989. Selection of stabletransformants can be performed using methods known in the art, such as,for example, resistance to cytotoxic drugs. Kaufman et al., Meth. inEnzymology 185:487-511, 1990, describes several selection schemes, suchas dihydrofolate reductase (DHFR) resistance. A suitable host strain forDHFR selection can be CHO strain DX-B11, which is deficient in DHFR(Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980). Aplasmid expressing the DHFR cDNA can be introduced into strain DX-B11,and only cells that contain the plasmid can grow in the appropriateselective media. Other examples of selectable markers that can beincorporated into an expression vector include cDNAs conferringresistance to antibiotics, such as G418 and hygromycin B. Cellsharboring the vector can be selected on the basis of resistance to thesecompounds.

Transcriptional and translational control sequences for mammalian hostcell expression vectors can be excised from viral genomes. Commonly usedpromoter sequences and enhancer sequences are derived from polyomavirus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites can be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment, which can also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978; Kaufman, Meth. inEnzymology, 1990). Smaller or larger SV40 fragments can also be used,provided the approximately 250 bp sequence extending from the Hind IIIsite toward the BglI site located in the SV40 viral origin ofreplication site is included.

Additional control sequences shown to improve expression of heterologousgenes from mammalian expression vectors include such elements as theexpression augmenting sequence element (EASE) derived from CHO cells(Morris et al., Animal Cell Technology, 1997, pp. 529-534) and thetripartite leader (TPL) and VA gene RNAs from Adenovirus 2 (Gingeras etal., J. Biol. Chem. 257:13475-13491, 1982). The internal ribosome entrysite (IRES) sequences of viral origin allows dicistronic mRNAs to betranslated efficiently (Oh and Sarnow, Current Opinion in Genetics andDevelopment 3:295-300, 1993; Ramesh et al., Nucleic Acids Research24:2697-2700, 1996). Expression of a heterologous cDNA as part of adicistronic mRNA followed by the gene for a selectable marker (e.g.DHFR) has been shown to improve transfectability of the host andexpression of the heterologous cDNA (Kaufman, Meth. in Enzymology,1990). Exemplary expression vectors that employ dicistronic mRNAs arepTR-DC/GFP described by Mosser et al., Biotechniques 22:150-161, 1997,and p2A5I described by Morris et al., Animal Cell Technology, 1997, pp.529-534.

A useful high expression vector, pCAVNOT, has been described by Mosleyet al., Cell 59:335-348, 1989. Other expression vectors for use inmammalian host cells can be constructed as disclosed by Okayama and Berg(Mol. Cell. Biol. 3:280, 1983). A useful system for stable high levelexpression of mammalian cDNAs in C127 murine mammary epithelial cellscan be constructed substantially as described by Cosman et al. (Mol.Immunol. 23:935, 1986). A useful high expression vector, PMLSV N1/N4,described by Cosman et al., Nature 312:768, 1984, has been deposited asATCC 39890. Additional useful mammalian expression vectors are describedin EP-A-0367566, and in U.S. patent application Ser. No. 07/701,415,filed May 16, 1991, incorporated by reference herein. The vectors can bederived from retroviruses. In place of the native signal sequence, aheterologous signal sequence can be added, such as the signal sequencefor IL-7 described in U.S. Pat. No. 4,965,195; the signal sequence forIL-2 receptor described in Cosman et al., Nature 312:768 (1984); theIL-4 signal peptide described in EP 367,566; the type I IL-1 receptorsignal peptide described in U.S. Pat. No. 4,968,607; and the type IIIL-1 receptor signal peptide described in EP 460,846.

Another useful expression vector, pFLAG, can be used. Flag® technologyis centered on the fusion of a low molecular weight (1 kD), hydrophilic,Flag® marker peptide to the N-terminus of a recombinant proteinexpressed by the pFLAG-1™ Expression Vector (1) (obtained from IBIKodak).

Polypeptides of the Invention

As noted above, the present invention also includes isolated andpurified polypeptides. As used herein, the “polypeptides” of theinvention refers to a genus of polypeptides that further encompassesproteins having the amino acid sequence of SEQ ID NO:6, SEQ ID NO:8, orSEQ ID NO:13, as well as those proteins having a high degree ofsimilarity (at least 90% homology) with such amino acid sequences andwhich proteins are biologically active. In addition, polypeptides of theinvention refers to the gene products of the nucleotides of SEQ ID NO:5,SEQ ID NO:7, and SEQ ID NO:12.

Isolation and Purification

The term “isolated and purified” as used herein, means that thepolypeptides or fragments of the invention are essentially free ofassociation with other proteins or polypeptides, for example, as apurification product of recombinant host cell culture or as a purifiedproduct from a non-recombinant source. The term “substantially purified”as used herein, refers to a mixture that contains polypeptides orfragments of the invention and is essentially free of association withother proteins or polypeptides, but for the presence of known proteinsthat can be removed using a specific antibody. The term “purified”refers to either the “isolated and purified” form of polypeptides of theinvention or the “substantially purified” form of polypeptides of theinvention, as both are described herein.

An isolated and purified polypeptide according to the invention can beproduced by recombinant expression systems as described above orpurified from naturally occurring cells.

In one preferred embodiment, the expression of recombinant IL-1 epsilonpolypeptides can be accomplished utilizing fusions of sequences encodingIL-1 epsilon polypeptides to sequences encoding another polypeptide toaid in the purification of polypeptides of the invention. An example ofsuch a fusion is a fusion of sequences encoding an IL-1 epsilonpolypeptide to sequences encoding the product of the malE gene of thepMAL-c2 vector of New England Biolabs, Inc. Such a fusion allows foraffinity purification of the fusion protein, as well as separation ofthe maltose binding protein portion of the fusion protein from thepolypeptide of the invention after purification.

The insertion of DNA encoding the IL-1 epsilon polypeptide into thepMAL-c2 vector can be accomplished in a variety of ways using knownmolecular biology techniques. The preferred construction of theinsertion contains a termination codon adjoining the carboxyl terminalcodon of the polypeptide of the invention. In addition, the preferredconstruction of the insertion results in the fusion of the aminoterminus of the polypeptide of the invention directly to the carboxylterminus of the Factor Xa cleavage site in the pMAL-c2 vector. A DNAfragment can be generated by PCR using DNA of the invention as thetemplate DNA and two oligonucleotide primers. Use of the oligonucleotideprimers generates a blunt-ended fragment of DNA that can be isolated byconventional means. This PCR product can be ligated together withpMAL-p2 (digested with the restriction endonuclease Xmn I) usingconventional means. Positive clones can be identified by conventionalmeans. Induction of expression and purification of the fusion proteincan be performed as per the manufacturer's instructions and as notedabove. This construction facilitates a precise separation of thepolypeptide of the invention from the fused maltose binding proteinutilizing a simple protease treatment as per the manufacturer'sinstructions. In this manner, purified IL-1 epsilon polypeptide can beobtained. Furthermore, such a constructed vector can be easily modifiedusing known molecular biology techniques to generate additional fusionproteins. It is understood, of course, that many different vectors andtechniques, as noted above, can be used for the expression andpurification of polypeptides of the invention and that this embodimentin no way limits the scope of the invention.

Recombinant protein produced in bacterial culture is usually isolated byinitial disruption of the host cells by any convenient method (includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents), centrifugation, extraction from cell pellets if aninsoluble polypeptide, or from the supernatant fluid if a solublepolypeptide, followed by one or more concentration, salting-out, ionexchange, affinity purification or size exclusion chromatography steps.As is known to the skilled artisan, procedures for purifying arecombinant protein will vary according to such factors as the type ofhost cells employed and whether or not the recombinant protein issecreted into the culture medium. For example, when expression systemsthat secrete the recombinant protein are employed, the culture mediumfirst can be concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. Following the concentration step, the concentratecan be applied to a purification matrix such as a gel filtration medium.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Sulfopropyl groups are preferred. Finally, one or morereversed-phase high performance liquid chromatography (RP-HPLC) stepsemploying hydrophobic RP-HPLC media, (e.g., silica gel having pendantmethyl or other aliphatic groups) can be employed to further purify thepolypeptides. Some or all of the foregoing purification steps, invarious combinations, are well known and can be employed to provide anisolated and purified recombinant protein.

It is also possible to utilize an affinity column comprising apolypeptide-binding protein of the invention, such as a monoclonalantibody generated against polypeptides of the invention, toaffinity-purify expressed polypeptides. These polypeptides can beremoved from an affinity column using conventional techniques, e.g., ina high salt elution buffer and then dialyzed into a lower salt bufferfor use or by changing pH or other components depending on the affinitymatrix utilized.

In this aspect of the invention, polypeptide-binding proteins, such asthe anti-polypeptide antibodies of the invention or other proteins thatmay interact with the polypeptide of the invention, can be bound to asolid phase support such as a column chromatography matrix or a similarsubstrate suitable for identifying, separating, or purifying cells thatexpress polypeptides of the invention on their surface. Adherence ofpolypeptide-binding proteins of the invention to a solid phasecontacting surface can be accomplished by any means, for example,magnetic microspheres can be coated with these polypeptide-bindingproteins and held in the incubation vessel through a magnetic field.Suspensions of cell mixtures are contacted with the solid phase that hassuch polypeptide-binding proteins thereon. Cells having polypeptides ofthe invention on their surface bind to the fixed polypeptide-bindingprotein and unbound cells then are washed away. This affinity-bindingmethod is useful for purifying, screening, or separating suchpolypeptide-expressing cells from solution. Methods of releasingpositively selected cells from the solid phase are known in the art andencompass, for example, the use of enzymes. Such enzymes are preferablynon-toxic and non-injurious to the cells and are preferably directed tocleaving the cell-surface binding partner.

Alternatively, mixtures of cells suspected of containingpolypeptide-expressing cells of the invention first can be incubatedwith a biotinylated polypeptide-binding protein of the invention.Incubation periods are typically at least one hour in duration to ensuresufficient binding to polypeptides of the invention. The resultingmixture then is passed through a column packed with avidin-coated beads,whereby the high affinity of biotin for avidin provides the binding ofthe polypeptide-binding cells to the beads. Use of avidin-coated beadsis known in the art. See Berenson, et al. J. Cell. Biochem., 10D:239(1986). Wash of unbound material and the release of the bound cells isperformed using conventional methods.

In the methods described above, suitable polypeptide-binding proteinsare anti-polypeptide antibodies, and other proteins that are capable ofhigh-affinity binding of polypeptides of the invention. A preferredpolypeptide-binding protein is an anti-polypeptide monoclonal antibody.

In a preferred embodiment, transformed yeast host cells are employed toexpress polypeptides of the invention as a secreted polypeptide in orderto simplify purification. Secreted recombinant polypeptide from a yeasthost cell fermentation can be purified by methods analogous to thosedisclosed by Urdal et al. (J. Chromatog. 296:171, 1984) (relating to theuse of two sequential, reversed-phase HPLC steps for purification).

Variants

The invention also includes variants of the polypeptides of theinvention. A polypeptide “variant” as referred to herein means apolypeptide substantially homologous to native polypeptides of theinvention, but which has an amino acid sequence different from that ofnative polypeptides (human, murine or other mammalian species) of theinvention because of one or more deletions, insertions or substitutions.The variant amino acid sequence preferably is at least 80% identical toa native polypeptide amino acid sequence. Also contemplated areembodiments in which a polypeptide or fragment comprises an amino acidsequence that is at least 90% identical, at least 95% identical, atleast 98% identical, at least 99% identical, or at least 99.9% identicalto the preferred polypeptide or fragment thereof. The percent identitycan be determined, for example, by comparing sequence information usingthe GAP computer program, version 6.0 described by Devereux et al.(Nucl. Acids Res. 12:387, 1984) and available from the University ofWisconsin Genetics Computer Group (UWGCG). The GAP program utilizes thealignment method of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970),as revised by Smith and Waterman (Adv. Appl. Math 2:482, 1981). Thepreferred default parameters for the GAP program include: (1) a unarycomparison matrix (containing a value of 1 for identities and 0 fornon-identities) for nucleotides, and the weighted comparison matrix ofGribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described bySchwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure,National Biomedical Research Foundation, pp. 353-358, 1979; (2) apenalty of 3.0 for each gap and an additional 0.10 penalty for eachsymbol in each gap; and (3) no penalty for end gaps.

Variants can comprise conservatively substituted sequences, meaning thata given amino acid residue is replaced by a residue having similarphysiochemical characteristics. Examples of conservative substitutionsinclude substitution of one aliphatic residue for another, such as Ile,Val, Leu, or Ala for one another, or substitutions of one polar residuefor another, such as between Lys and Arg; Glu and Asp; or Gln and Asn.Other such conservative substitutions, for example, substitutions ofentire regions having similar hydrophobicity characteristics, are wellknown. Naturally occurring variants are also encompassed by theinvention. Examples of such variants are proteins that result fromalternate mRNA splicing events, proteolytic cleavage of the IL-1 epsilonpolypeptides, or transcription/translation from different alleles.Variations attributable to proteolysis include, for example, differencesin the N or C-termini upon expression in different types of host cells,due to proteolytic removal of one or more terminal amino acids from thepolypeptides (generally from 1-5 terminal amino acids) of the invention.

Oligomers

The polypeptides of the invention can also exist as oligomers, such ascovalently linked or non-covalently linked dimers or trimers. Oligomerscan be linked by disulfide bonds formed between cysteine residues ondifferent polypeptides.

In one embodiment of the invention, a polypeptide dimer is created byfusing polypeptides of the invention to the Fc region of an antibody(e.g., IgG1) in a manner that does not interfere with the biologicalactivity of these polypeptides. The Fc region preferably is fused to theC-terminus of a soluble polypeptide of the invention, to form an Fcfusion or an Fc polypeptide. The terms “Fc fusion protein” or “Fcpolypeptides” as used herein includes native and mutein forms, as wellas truncated Fc polypeptides containing the hinge region that promotesdimerization. Exemplary methods of making Fc polypeptides set forthabove are disclosed in U.S. Pat. Nos. 5,426,048 and 5,783,672, both ofwhich are incorporated herein by reference.

General preparation of fusion proteins comprising heterologouspolypeptides fused to various portions of antibody-derived polypeptides(including the Fc domain) has been described, e.g., by Ashkenazi et al.(PNAS USA 88:10535, 1991) and Byrn et al. (Nature 344:677, 1990), herebyincorporated by reference. A gene fusion encoding the polypeptide:Fcfusion protein of the invention is inserted into an appropriateexpression vector. Polypeptide:Fc fusion proteins are allowed toassemble much like antibody molecules, whereupon interchain disulfidebonds form between Fc polypeptides, yielding divalent polypeptides ofthe invention. If fusion proteins are made with both heavy and lightchains of an antibody, it is possible to form a polypeptide oligomerwith as many as four polypeptides extracellular regions. Alternatively,one can link two soluble polypeptide domains with a peptide linker.

Alterations

As stated above, the invention provides isolated and purifiedpolypeptides, and fragments thereof, both recombinant andnon-recombinant. Variants and derivatives of native polypeptides can beobtained by mutations of nucleotide sequences coding for nativepolypeptides. Alterations of the native amino acid sequence can beaccomplished by any of a number of conventional methods. Mutations canbe introduced at particular loci by synthesizing oligonucleotidescontaining a mutant sequence, flanked by restriction sites enablingligation to fragments of the native sequence. Following ligation, theresulting reconstructed sequence encodes an analog having the desiredamino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene whereinpredetermined codons can be altered by substitution, deletion orinsertion. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981);Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methodsin Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462,all of which are incorporated by reference.

Polypeptides of the invention can also be modified to create polypeptidederivatives by forming covalent or aggregative conjugates with otherchemical moieties, such as glycosyl groups, polyethylene glycol (PEG)groups, lipids, phosphate, acetyl groups and the like. Covalentderivatives of polypeptides of the invention can be prepared by linkingthe chemical moieties to functional groups on polypeptide amino acidside chains or at the N-terminus or C-terminus of a polypeptide of theinvention or the extracellular domain thereof. Other derivatives ofpolypeptides within the scope of this invention include covalent oraggregative conjugates of these polypeptides or peptide fragments withother proteins or polypeptides, such as by synthesis in recombinantculture as N-terminal or C-terminal fusions. For example, the conjugatecan comprise a signal or leader polypeptide sequence (e.g. thealpha-factor leader of Saccharomyces) at the N-terminus of a polypeptideof the invention. The signal or leader peptide co-translationally orpost-translationally directs transfer of the conjugate from its site ofsynthesis to a site inside or outside of the cell membrane or cell wall.

Polypeptide conjugates can also comprise peptides added to facilitatepurification and identification of polypeptides of the invention. Suchpeptides include, for example, poly-His or the antigenic identificationpeptides described in U.S. Pat. No. 5,011,912 and in Hopp et al.,Bio/Technology 6:1204, 1988.

The invention further includes polypeptides of the invention with orwithout associated native-pattern glycosylation. Polypeptides expressedin yeast or mammalian expression systems (e.g., COS-1 or COS-7 cells)can be similar to or significantly different from a native polypeptidein molecular weight and glycosylation pattern, depending upon the choiceof expression system. Expression of polypeptides of the invention inbacterial expression systems, such as E. coli, provides non-glycosylatedmolecules. Glycosyl groups can be removed through conventional methods,in particular those utilizing glycopeptidase. In general, glycosylatedpolypeptides of the invention can be incubated with a molar excess ofglycopeptidase (Boehringer Mannheim).

Correspondingly, equivalent DNA constructs that encode various additionsor substitutions of amino acid residues or sequences, or deletions ofterminal or internal residues or sequences are encompassed by theinvention. For example, N-glycosylation sites in the polypeptideextracellular domain can be modified to preclude glycosylation, allowingexpression of a reduced carbohydrate analog in mammalian and yeastexpression systems. N-glycosylation sites in eukaryotic polypeptides arecharacterized by an amino acid triplet Asn-X-Y, wherein X is any aminoacid except Pro and Y is Ser or Thr. Appropriate substitutions,additions, or deletions to the nucleotide sequence encoding thesetriplets will result in prevention of attachment of carbohydrateresidues at the Asn side chain. Alteration of a single nucleotide,chosen so that Asn is replaced by a different amino acid, for example,is sufficient to inactivate an N-glycosylation site. Known proceduresfor inactivating N-glycosylation sites in proteins include thosedescribed in U.S. Pat. No. 5,071,972 and EP 276,846, hereby incorporatedby reference.

In another example, sequences encoding Cys residues that are notessential for biological activity can be altered to cause the Cysresidues to be deleted or replaced with other amino acids, preventingformation of incorrect intramolecular disulfide bridges uponrenaturation. Other equivalents are prepared by modification of adjacentdibasic amino acid residues to enhance expression in yeast systems inwhich KEX2 protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding, or substituting residues to alter Arg-Arg, Arg-Lys,and Lys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Lys-Lys pairings are considerably less susceptible to KEX2cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents aconservative and preferred approach to inactivating KEX2 sites.

Fragments and Uses Thereof

In yet another aspect of the invention, the polypeptides of theinvention can be subjected to fragmentation into peptides by chemicaland enzymatic means.

Although all methods of fragmentation are encompassed by the invention,chemical fragmentation is a preferred embodiment, and includes the useof cyanogen bromide to cleave under neutral or acidic conditions suchthat specific cleavage occurs at methionine residues (E. Gross, Methodsin Enz. 11:238-255, 1967). This can further include additional steps,such as a carboxymethylation step to convert cysteine residues to anunreactive species.

Enzymatic fragmentation is another preferred embodiment, and includesthe use of a protease such as Asparaginylendo-peptidase,Arginylendo-peptidase, Achromobacter protease I, Trypsin, Staphlococcusaureus V8 protease, Endoproteinase Asp-N, or Endoproteinase Lys-C underconventional conditions to result in cleavage at specific amino acidresidues. Asparaginylendo-peptidase can cleave specifically on thecarboxyl side of the asparagine residues present within the polypeptidesof the invention. Arginylendo-peptidase can cleave specifically on thecarboxyl side of the arginine residues present within thesepolypeptides. Achromobacter protease I can cleave specifically on thecarboxyl side of the lysine residues present within the polypeptides(Sakiyama and Nakat, U.S. Pat. No. 5,248,599; T. Masaki et al., Biochim.Biophys. Acta 660:44-50, 1981; T. Masaki et al., Biochim. Biophys. Acta660:51-55, 1981). Trypsin can cleave specifically on the carboxyl sideof the arginine and lysine residues present within polypeptides of theinvention. Enzymatic fragmentation may also occur with a protease thatcleaves at multiple amino acid residues. For example, Staphlococcusaureus V8 protease can cleave specifically on the carboxyl side of theaspartic and glutamic acid residues present within polypeptides (D. W.Cleveland, J. Biol. Chem. 3:1102-1106, 1977). Endoproteinase Asp-N cancleave specifically on the amino side of the asparagine residues presentwithin polypeptides. Endoproteinase Lys-C can cleave specifically on thecarboxyl side of the lysine residues present within polypeptides of theinvention. Other enzymatic and chemical treatments can likewise be usedto specifically fragment these polypeptides into a unique set ofspecific peptides.

Of course, the peptides and fragments of the polypeptides of theinvention can also be produced by conventional recombinant processes andsynthetic processes well known in the art. With regard to recombinantprocesses, the polypeptides and peptide fragments encompassed byinvention can have variable molecular weights, depending upon the hostcell in which they are expressed. Glycosylation of polypeptides andpeptide fragments of the invention in various cell types can result invariations of the molecular weight of these pieces, depending upon theextent of modification. Consistent polypeptides and peptide fragmentscan be obtained by pretreating with N-glycanase to remove glycosylation,or expressing the polypeptides in bacterial hosts.

The polypeptides and fragments thereof can also be varied by fusingadditional peptide sequences to either or both the amino and carboxylterminal ends of polypeptides of the invention. Fusions of additionalpeptide sequences at the amino and carboxyl terminal ends ofpolypeptides of the invention can be used to enhance expression of thesepolypeptides or aid in the purification of the protein. Of course,mutations can be introduced into polypeptides of the invention usingroutine and known techniques of molecular biology. For example, amutation can be designed so as to eliminate a site of proteolyticcleavage by a specific enzyme or a site of cleavage by a specificchemically induced fragmentation procedure.

Finally, as to the kits that are encompassed by the invention, theconstituents of such kits can be varied, but typically contain thepolypeptide and fragments thereof. Kits can further contain antibodiesdirected against polypeptides or fragments thereof of the invention.

Sense and Antisense Oligonucleotides

In yet another embodiment of the invention, antisense or senseoligonucleotides comprising a single-stranded nucleic acid sequence(either RNA or DNA) capable of binding to a target mRNA sequence(forming a duplex) or to the sequence in the double-stranded DNA helix(forming a triple helix) can be made according to the invention.Antisense or sense oligonucleotides, according to the present invention,comprise a fragment of the coding region of cDNA (SEQ ID NO:5, SEQ IDNO:7, or SEQ ID NO:12). Such a fragment generally comprises at leastabout 14 nucleotides, preferably from about 14 to about 30 nucleotides.The ability to create an antisense or a sense oligonucleotide, basedupon a cDNA sequence for a given protein is described in, for example,Stein and Cohen, Cancer Res. 48:2659, 1988 and van der Krol et al.,BioTechniques 6:958, 1988.

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of complexes that block translation(RNA) or transcription (DNA) by one of several means, including enhanceddegradation of the duplexes, premature termination of transcription ortranslation, or by other means. The antisense oligonucleotides thus canbe used to block expression of polypeptides of the invention. Antisenseor sense oligonucleotides further comprise oligonucleotides havingmodified sugar-phosphodiester backbones (or other sugar linkages, suchas those described in WO 91/06629) and wherein such sugar linkages areresistant to endogenous nucleases. Such oligonucleotides with resistantsugar linkages are stable in vivo (i.e., capable of resisting enzymaticdegradation), but retain sequence specificity to be able to bind totarget nucleotide sequences. Other examples of sense or antisenseoligonucleotides include those oligonucleotides that are covalentlylinked to organic moieties, such as those described in WO 90/10448, andother moieties that increase affinity of the oligonucleotide for atarget nucleic acid sequence, such as poly-(L-lysine). Further still,intercalating agents, such as ellipticine, and alkylating agents ormetal complexes can be attached to sense or antisense oligonucleotidesto modify binding specificities of the antisense or senseoligonucleotide for the target nucleotide sequence.

Antisense or sense oligonucleotides can be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. Antisense or sense oligonucleotides are preferably introducedinto a cell containing the target nucleic acid sequence by insertion ofthe antisense or sense oligonucleotide into a suitable retroviralvector, then contacting the cell with the retrovirus vector containingthe inserted sequence, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, the murine retrovirus M-MuLV,N2 (a retrovirus derived from M-MuLV), or the double copy vectorsdesignated DCT5A, DCT5B and DCT5C (see PCT Application US 90/02656).

Alternatively, sense or antisense oligonucleotides also can beintroduced into a cell containing the target nucleotide sequence byformation of a conjugate with a ligand binding molecule, as described inWO 91/04753. Suitable ligand binding molecules include, but are notlimited to, cell surface receptors, growth factors, other cytokines, orother ligands that bind to cell surface receptors. Preferably,conjugation of the ligand binding molecule does not substantiallyinterfere with the ability of the ligand binding molecule to bind to itscorresponding molecule or receptor, or block entry of the sense orantisense oligonucleotide or its conjugated version into the cell.

In yet another embodiment, a sense or an antisense oligonucleotide canbe introduced into a cell containing the target nucleic acid sequence byformation of an oligonucleotide-lipid complex, as described in WO90/10448. The sense or antisense oligonucleotide-lipid complex ispreferably dissociated within the cell by an endogenous lipase.

Chromosome Mapping

In still another embodiment, oligonucleotides representing all or aportion of SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:12 can be used bythose skilled in the art using well-known techniques to identify thehuman chromosome 2, and the specific locus thereof, that contains theDNA of IL-1 family members, for example, IL-1 epsilon. As set forthbelow, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:12 have been mapped byradiation hybrid mapping to the long arm (2q) region of chromosome 2.That region is associated with specific diseases which include but arenot limited to glaucoma, ectodermal dysplasia, insulin-dependentdiabetes mellitus, wrinkly skin syndrome, T-cell leukemia/lymphoma,asthma, and tibial muscular dystrophy. Thus, SEQ ID NO:5, SEQ ID NO:7,SEQ ID NO:12, or a fragment of these sequences can be used by oneskilled in the art using well-known techniques to study the abovedescribed diseases and other abnormalities relating to chromosome 2.This would enable one to distinguish conditions in which this marker isrearranged or deleted. In addition, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:12, or a fragment thereof can be used as a positional marker to mapother human genes of unknown location.

Therapeutic and Research Uses

Another embodiment of the invention relates to therapeutic uses of IL-1epsilon. IL-1 ligands play a central role in protection againstinfection and in promoting immune and inflammatory responses, whichincludes cellular signal transduction, activating vascular endothelialcells and lymphocytes, induction of inflammatory cytokines, acute phaseproteins, hematopoiesis, fever, bone resorption, prostaglandins,metalloproteinases, and adhesion molecules. With the continued increasein the number of known IL-1 family members, a suitable classificationscheme is one based on comparing polypeptide structure as well asfunction (activation and regulatory properties). Thus, IL-1 epsilon,like IL-1 alpha, IL-1 beta, and IL-18, would likely be involved in manyof the functions noted above as well as promote inflammatory responsesand therefore perhaps be involved in the causation and maintenance ofinflammatory and/or autoimmune diseases such as rheumatoid arthritis(and/or other arthritic conditions that have an inflammatory orautoimmune component, for example, ankylosing spondylitis), inflammatorybowel disease (including Crohn's Disease and ulcerative colitis), andpsoriasis (including psoriatic arthritis). Other inflammatory and/orautoimmune diseases in which IL-1 epsilon?????? may be implicatedinclude asthma (and other pulmonary conditions relating to an immune orinflammatory response and/or in which airway hyperreactivity plays arole), and multiple sclerosis (and/or other demyelinating conditionsthat have an inflammatory or autoimmune component). As such, alterationsin the expression and/or activation of IL-1 family members such as IL-1epsilon can have profound effects on a plethora of cellular processes,including, but not limited to, activation or inhibition of cell specificresponses, proliferation, and inflammatory reactions based on changes insignal transduction.

Accordingly, IL-1 epsilon has therapeutic uses, such as protectingagainst infection and generating immune and inflammatory responses inindividuals whose immune and inflammatory responses are inappropriate ornonresponsive. For example, IL-1 epsilon may be useful in stimulatingthe immune system of individuals whose immune system isimmunosuppressed. Similarly, because IL-1 epsilon likely promotesinflammatory responses and is involved in the causation and maintenanceof inflammatory and/or autoimmune diseases, antagonists of IL-1 epsilonare useful in inhibiting or treating inflammatory and/or automimmunedisease. Thus, antagonists of IL-1 epsilon will be useful in treating asrheumatoid arthritis, inflammatory bowel disease, and psoriasis.

IL-1 mediated cellular signaling often involves a molecular activationcascade, during which a receptor propagates a ligand-receptor mediatedsignal by specifically activating intracellular kinases whichphosphorylate target substrates, resulting in the activation of thetranscription factor NFkappaB and the protein kinases Jun N-terminalkinase and p38 map kinase. These substrates can themselves be kinaseswhich become activated following phosphorylation. Alternatively, theycan be adaptor molecules that facilitate down-stream signaling throughprotein-protein interaction following phosphorylation.

Given the data presented in Example III, below, it is likely that IL-1epsilon is an agonist, such as, for example IL-1 alpha or IL-18. Asstated above, such agonists are useful in promoting immune andinflammatory responses in individuals whose own immune systems areinappropriately under responsive. Antagonists of IL-1 epsilon will beuseful in treating or ameliorating conditions in which the immune and/orinflammatory response is over responsive. For purposes of antagonizingIL-1 epsilon activity, inhibitors of IL-1 epsilon can be engineered ordesigned using techniques known in the art.

Antagonists of IL-1 epsilon will be useful in treating arthriticconditions that have an inflammatory or autoimmune component, forexample, rheumatoid arthritis and/or ankylosing spondylitis;inflammatory bowel disease, including Crohn's Disease and ulcerativecolitis, and psoriasis (including psoriatic arthritis). Otherinflammatory and/or autoimmune diseases in which IL-1 epsilon isimplicated include pulmonary conditions relating to an immune orinflammatory response and/or in which airway hyperreactivity plays arole, for example, asthma, infection-associated airway hyperactivity,granulomatous lung disease, emphysema and chronic fibrosing alveolitisand acute hyperoxic lung damage. and demyelinating conditions that havean inflammatory or autoimmune component, for example, multiple sclerosisand/or chronic inflammatory demyelinating polyneuropathy. Antagonists ofIL-1 epsilon will also be useful in ameliorating these conditions.

Additional conditions for which an autoimmune and/or inflammatorycomponent is a contributory factor (and thus, for which antagonists ofIL-1 epsilon are useful) include cardiovascular conditions such asstroke, acute myocardial infarction, unstable angina, arterialrestenosis and congestive heart failure. IL-1 epsilon antagonists areuseful in treating or preventing osteoporosis and/or osteoarthritis, aswell as glomerulonephritis, uveitis, and/or Behcet's syndrome. Anautoimmune or inflammatory component also plays a role in the cause ormaintenance of sepsis, acute pancreatitis, diabetes (particularly TypeII, insulin dependent diabetes), endometriosis, and periodontal disease.Similarly, the inflammatory response causes or exacerbates heat strokeand glaucoma, and the cytokines involved in the immune/inflammatoryresponse play a supportive role in neoplastic disease (for example, inmultiple myeloma and/or myeloid leukemia), facilitating the growth ofneoplastic cells. Accordingly, antagonists of IL-1 epsilon are useful intreating or ameliorating these conditions by downregulating the immuneand/or inflammatory response that plays a causative role therein.

The compositions of the present invention, including IL-1 epsiloninhibitors, can be introduced into the extracellular environment bywell-known means, such as by administering the protein intravenously orby coupling it to a monoclonal antibody targeted to a specific celltype, to thereby affect signaling. When used as a therapeutic agent,polypeptides of the invention can be formulated into pharmaceuticalcompositions according to known methods. The compositions can becombined in admixture, either as the sole active material or with otherknown active materials, with pharmaceutically suitable diluents (e.g.,Tris-HCl, acetate, phosphate), preservatives (e.g., Thimerosal, benzylalcohol, parabens), emulsifiers, solubilizers, adjuvants and/orcarriers. Suitable carriers and their formulations are described inRemington's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Co.In addition, such compositions can contain polypeptides complexed withpolyethylene glycol (PEG), metal ions, or incorporated into polymericcompounds such as polyacetic acid, polyglycolic acid, hydrogels, etc.,or incorporated into liposomes, microemulsions, micelles, unilamellar ormultilamellar vesicles, erythrocyte ghosts or spheroblasts. Suchcompositions will influence the physical state, solubility, stability,rate of in vivo release, and rate of in vivo clearance of polypeptidesof the invention.

The dosage of the composition can be readily determined by those ofordinary skill in the art. The amount to be administered and thefrequency of administration can be determined empirically and will takeinto consideration the age and size of the patient being treated, aswell as the malady being treated.

Treatment comprises administering the composition by any method familiarto those of ordinary skill in the art, including intravenous,intraperitoneal, intracorporeal injection, intra-articular,intraventricular, intrathecal, intramuscular, subcutaneous, topically,tonsillar, intranasally, intravaginally, and orally. The composition mayalso be given locally, such as by injection into the particular area,either intramuscularly or subcutaneously.

In addition, the DNA, polypeptides, and antibodies against polypeptidesof the invention can be used as reagents in a variety of researchprotocols. A sample of such research protocols are given in Sambrook etal. Molecular Cloning: A Laboratory Manual, 2^(nd) ed. Vol. 1-3, ColdSpring Harbor Laboratory Press, (1989). For example, these reagents canserve as markers for cell-specific or tissue-specific expression of RNAor proteins. Similarly, these reagents can be used to investigateconstitutive and transient expression of RNA or polypeptides. As notedabove, the DNA can be used to determine the chromosomal location of DNAand to map genes in relation to this chromosomal location. The DNA canalso be used to examine genetic heterogeneity and heredity through theuse of techniques such as genetic fingerprinting, as well as to identifyrisks associated with genetic disorders. The DNA can be further used toidentify additional genes related to the DNA and to establishevolutionary trees based on the comparison of sequences. The DNA andpolypeptides can be used to select for those genes or proteins that arehomologous to the DNA or polypeptides, through positive screeningprocedures such as Southern blotting and immunoblotting and throughnegative screening procedures such as subtraction.

Further, because IL-1 epsilon is a ligand, it takes part inprotein-protein interactions with at least one or more proteins, i.e.its receptor(s). Thus, the polypeptides and fragments of the inventioncan be used as reagents to identify (a) proteins that the polypeptideregulates, and (b) proteins with which it might interact. Therefore,IL-1 epsilon ligands or polypeptides comprising portions of an IL-1epsilon ligand could be used by coupling recombinant protein to anaffinity matrix, or by using them as “baits” in the yeast 2-hybridsystem according to well established molecular biology techniques, toidentify proteins that interact directly with the polypeptide of theinvention. Further, the IL-1 epsilon polypeptides and fragments of thepresent invention find use in studies directed toward discovering IL-1receptors and/or IL-1 epsilon receptors. For example, IL-1 epsilonpolypeptides and IL-1 epsilon polypeptide fragments can be used inbinding studies to identify receptor-expressing cells. Suitable bindingstudies are known in the art and are well within the knowledge of thoseskilled in the art. Similarly, the IL-1 epsilon polypeptides andpolypeptide fragments of the present invention find additional uses incloning receptors using expression cloning techniques.

The polypeptides and fragments thereof can also be used as reagents inthe study of signaling pathways utilized by IL-1 and IL-1R homologs orfamily members, and/or in blocking those signaling pathways. Such novelIL-1 receptor homologs can be specifically used as reagents to identifynovel molecules involved in signal transduction pathways, characterizecell and tissue expression, understand their roles in development,immune, and inflammatory responses, and identify regulatory moleculesand physiologically relevant protein substrates.

Alternatively, polypeptides of the invention could be engineered priorto expression with a tag such as poly-His or Flag®, then be expressedand purified using either nickel chelate chromatography or anti-Flag®antibody coupled to a resin, respectively. Once bound to the resin, thepolypeptide of the invention could be covalently attached using abifunctional cross-linking agent using well established techniques. Thecovalently bound polypeptide to the resin could then be used to purifymolecules from cell lysates or cell supernatants (following treatmentwith various reagent) through their affinity for the polypeptide of theinvention.

Antibodies

Antibodies that are immunoreactive with the polypeptides of theinvention are provided herein. Such antibodies specifically bind to thepolypeptides via the antigen-binding sites of the antibody (as opposedto non-specific binding). Thus, the polypeptides, fragments, variants,fusion proteins, etc., as set forth above may be employed as“immunogens” in producing antibodies immunoreactive therewith. Morespecifically, the polypeptides, fragment, variants, fusion proteins,etc. contain antigenic determinants or epitopes that elicit theformation of antibodies.

These antigenic determinants or epitopes can be either linear orconformational (discontinuous). Linear epitopes are composed of a singlesection of amino acids of the polypeptide, while conformational ordiscontinuous epitopes are composed of amino acids sections fromdifferent regions of the polypeptide chain that are brought into closeproximity upon protein folding (C. A. Janeway, Jr. and P. Travers,Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Becausefolded proteins have complex surfaces, the number of epitopes availableis quite numerous; however, due to the conformation of the protein andsteric hindrances, the number of antibodies that actually bind to theepitopes is less than the number of available epitopes (C. A. Janeway,Jr. and P. Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nded. 1996)). Epitopes may be identified by any of the methods known inthe art.

Thus, one aspect of the present invention relates to the antigenicepitopes of the polypeptides of the invention. Such epitopes are usefulfor raising antibodies, in particular monoclonal antibodies, asdescribed in more detail below. Additionally, epitopes from thepolypeptides of the invention can be used as research reagents, inassays, and to purify specific binding antibodies from substances suchas polyclonal sera or supernatants from cultured hybridomas. Suchepitopes or variants thereof can be produced using techniques well knownin the art such as solid-phase synthesis, chemical or enzymatic cleavageof a polypeptide, or using recombinant DNA technology.

As to the antibodies that can be elicited by the epitopes of thepolypeptides of the invention, whether the epitopes have been isolatedor remain part of the polypeptides, both polyclonal and monoclonalantibodies may be prepared by conventional techniques. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).

Hybridoma cell lines that produce monoclonal antibodies specific for thepolypeptides of the invention are also contemplated herein. Suchhybridomas may be produced and identified by conventional techniques.One method for producing such a hybridoma cell line comprises immunizingan animal with a polypeptide or a DNA encoding a polypeptide; harvestingspleen cells from the immunized animal; fusing said spleen cells to amyeloma cell line, thereby generating hybridoma cells; and identifying ahybridoma cell line that produces a monoclonal antibody that binds thepolypeptide. The monoclonal antibodies may be recovered by conventionaltechniques.

The monoclonal antibodies of the present invention include chimericantibodies, e.g., humanized versions of murine monoclonal antibodies.Such humanized antibodies may be prepared by known techniques and offerthe advantage of reduced immunogenicity when the antibodies areadministered to humans. In one embodiment, a humanized monoclonalantibody comprises the variable region of a murine antibody (or just theantigen binding site thereof) and a constant region derived from a humanantibody. Alternatively, a humanized antibody fragment may comprise theantigen binding site of a murine monoclonal antibody and a variableregion fragment (lacking the antigen-binding site) derived from a humanantibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in Riechmann etal. (Nature 332:323, 1988), Liu et al. (Proc. Natl. Acad. Sci. USA84:3439, 1987), Larrick et al. (Bio/Technology 7:934, 1989), and Winterand Harris (TIPS 14:139, May, 1993).

In addition to antibodies that can be produced via recombinant methods,human antibodies can be produced in animals that have been geneticallymanipulated to have human immunoglobulin genes (transgenic animals).Procedures to generate antibodies transgenically can be found in GB2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806 and related patentsclaiming priority therefrom, all of which are incorporated by referenceherein. Preferably, for use in humans, the antibodies are human orhumanized; techniques for creating such human or humanized antibodiesare also well known and are commercially available from, for example,Medarex Inc. (Princeton, N.J.) and Abgennix Inc. (Fremont, Calif.).

Antigen-binding fragments of the antibodies, which may be produced byconventional techniques, are also encompassed by the present invention.Examples of such fragments include, but are not limited to, Fab andF(ab′)2 fragments. Antibody fragments and derivatives produced bygenetic engineering techniques are also provided.

In one embodiment, the antibodies are specific for the polypeptides ofthe present invention and do not cross-react with other proteins.Screening procedures by which such antibodies may be identified are wellknown, and may involve immunoaffinity chromatography, for example.

The antibodies of the invention can be used in assays to detect thepresence of the polypeptides or fragments of the invention, either invitro or in vivo. The antibodies also may be employed in purifyingpolypeptides or fragments of the invention by immunoaffinitychromatography.

Drug Discovery

The purified polypeptides according to the invention will facilitate thediscovery of inhibitors (or antagonists) and/or agonists of suchpolypeptides. The use of a purified polypeptide of the invention in thescreening of potential inhibitors and/or agonists thereof is importantand can eliminate or reduce the possibility of interfering reactionswith contaminants.

In addition, polypeptides of the invention can be used forstructure-based design of polypeptide-inhibitors. Such structure-baseddesign is also known as “rational drug design.” The polypeptides can bethree-dimensionally analyzed by, for example, X-ray crystallography,nuclear magnetic resonance or homology modeling, all of which arewell-known methods. The use of the polypeptide structural information inmolecular modeling software systems to assist in inhibitor design andinhibitor-polypeptide interaction is also encompassed by the invention.Such computer-assisted modeling and drug design can utilize informationsuch as chemical conformational analysis, electrostatic potential of themolecules, protein folding, etc. For example, most of the design ofclass-specific inhibitors of metalloproteases has focused on attempts tochelate or bind the catalytic zinc atom. Synthetic inhibitors areusually designed to contain a negatively-charged moiety to which isattached a series of other groups designed to fit the specificitypockets of the particular protease. A particular method of the inventioncomprises analyzing the three dimensional structure of polypeptides ofthe invention for likely binding sites of substrates, synthesizing a newmolecule that incorporates a predictive reactive site, and assaying thenew molecule as described above.

Specific screening methods are known in the art and along withintegrated robotic systems and collections of chemical compounds/naturalproducts are extensively incorporated in high throughput screening sothat large numbers of test compounds can be tested for antagonist oragonist activity within a short amount of time. These methods includehomogeneous assay formats such as fluorescence resonance energytransfer, fluorescence polarization, time-resolved fluorescenceresonance energy transfer, scintillation proximity assays, reporter geneassays, fluorescence quenched enzyme substrate, chromogenic enzymesubstrate and electrochemiluminescence, as well as more traditionalheterogeneous assay formats such as enzyme-linked immunosorbant assays(ELISA) or radioimmunoassays. Homogeneous assays are preferred. Alsocomprehended herein are cell-based assays, for example those utilizingreporter genes, as well as functional assays that analyze the effect ofan antagonist or agonist on biological function(s) or activity(ies) ofIL-1 epsilon (for example, secretion of cytokines as disclosed herein).

Accordingly, in one aspect of the invention, there is provided a methodfor screening a test compound to determine whether the test compoundaffects a biological activity of an IL-1 epsilon polypeptide, the methodcomprising contacting the test compound and the IL-1 epsilon polypeptidewith cells capable of exhibiting the biological activity when contactedwith IL-1 epsilon, and analyzing the cells for the occurrence of thebiological activity, wherein if the biological activity observed in thepresence of the test compound differs from the biological activity thatis observed when the test compound is absent, the test compound affectsthe biological activity of the IL-1 epsilon.

As used herein, the IL-1 epsilon polypeptide comprises a polypeptideselected from the group consisting of the polypeptides of SEQ ID NO:6,SEQ ID NO:8, and SEQ ID NO:13, and polypeptides encoded by DNAs thathybridize under moderately stringent conditions to the DNAs of SEQ IDNO:5, SEQ ID NO:7, or SEQ ID NO:12. Such polypeptides includepolypeptides comprising variant amino acid sequences that are at least80% identical to the polypeptides of SEQ ID NO:6, SEQ ID NO:8, or SEQ IDNO:13 (preferably, the variant amino acid sequences that are at least90% identical, more preferably at least 95% identical, most preferablyat least 97% identical, to the polypeptides of SEQ ID NO:6, SEQ ID NO:8,or SEQ ID NO:13). Additional examples of useful IL-1 epsilonpolypeptides include polypeptides comprising the amino acid sequences ofSEQ ID NOs:6, 8, or 13 wherein the polypeptides comprise alterations tothe amino acid sequences selected from the group consisting ofinactivated N-glycosylation site(s), inactivated protease processingsite(s), conservative amino acid substitution(s), and combinationsthereof. Moreover, fragments of the aforesaid polypeptides that have atleast one activity of IL-1 epsilon as described below are alsocomprehended herein (for example, a fragment as shown in FIG. 4).

IL-1 epsilon biological activity includes, but is not limited to, IL-1epsilon induced cytokine expression, IL-1 epsilon induced expression ofmolecules indicative of activation of an immune or inflammatory response(for example, COX2, iNOS), IL-1 epsilon induced cell-surface moleculeexpression, activation of one or more signaling cascades, induction ofmRNAs for the aforementioned proteins, induction of cell proliferationand/or cell death, induction of morphological and/or functional changesin cells, and combinations thereof. The inventive methods comprisemethods of assaying for any of these biological activities. When themethods of the present invention include assaying for IL-1 epsiloninduced cytokine expression, cytokines that may be assayed include (butare not limited to) IL-1 alpha, IL-1 beta, TNF-alpha, IL-10, IFN-gamma,IL-12 (in particular, the p40 subunit), IL-6, IL-1ra, IL-4, IL-13,GM-CSF, IL-18, IL-1 homologs such as IL-1 delta, IL-1 eta, IL-1 theta,IL-1 zeta, and IL-1 H1, and combinations thereof. Similarly, when thescreening methods of the present invention include assaying for IL-1epsilon induced cell surface molecule expression, the cell surfacemolecules that may be assayed include ICAM-1, TLR4, TLR5, TLR9, DC-B7,MHC class I and II antigens, VCAM, ELAM, B7-1, B7-2, CD40L, andcombinations thereof.

IL-1 epsilon mediated activation of signaling pathways often involves acascade of molecular changes, for example as discussed previouslywherein a receptor propagates a ligand-receptor mediated signal byspecifically activating intracellular kinases which phosphorylate targetsubstrates (which can themselves be kinases that become activatedfollowing phosphorylation, or adaptor molecules that facilitatedown-stream signaling through protein-protein interaction followingphosphorylation), resulting in the activation of other factors (forexample, NFkappaB). When the screening methods of the present inventioninclude assaying for IL-1 epsilon induced activation of signalingpathways, the signaling pathways that may be assayed include thoseinvolving activation of NFkappaB. Assaying for activation signalingcascades further includes detecting phosphorylation of molecules thatoccurs during the signaling cascade, as in the phosphorylation ofIkappaB (including IkappaB degradation assays, and assays for freeIkappaB), p38 MAP kinase, and Stress-Activated Protein Kinase(SAPK/JNK).

Moreover, those of skill in the art understand that biologicalactivity(ies) is/are most often induced by the binding of a ligand(i.e., IL-1 epsilon) to a receptor (counterstructure or binding moiety)present on a cell; accordingly, as previously described, IL-1 epsilonpolypeptides (including IL-1 epsilon polypeptide fragments) can be usedin binding studies to identify receptor-expressing cells. Such bindingstudies also provide assays useful in the inventive methods. IL-1epsilon polypeptides may also be used to clone receptors (or othermolecules that bind IL-1 epsilon) and to screen for molecules that blockreceptor/ligand interactions. Those of ordinary skill in the art furtherunderstand that biological activities include cell proliferation, celldeath, and changes in cell morphology and/or function (for example,activation, maturation); assays that evaluate such effects of IL-1epsilon are known in the art, and will also be useful in the inventivemethods.

The inventive methods further encompass performing more than one assayto discover and/or analyze agonists or antagonists of IL-1 epsilonactivity (i.e., combination methods). Generally, such methods compriseselecting test compounds that affect a property of IL-1 epsilon (i.e.,an ability of IL-1 epsilon to bind an IL-1 epsilon counter structure),then testing the selected compounds for an effect on another property ofIL-1 epsilon (i.e., contacting the selected test compounds and an IL-1epsilon polypeptide with cells capable of exhibiting a biologicalactivity when contacted with IL-1 epsilon, and determining whether thecompounds affect the biological activity. For example, the inventivemethods may comprise a first assay to determine whether a candidatemolecule interacts with (binds to) IL-1 epsilon. Preferably, the firstassay is in a high throughput format, numerous forms of which are knownin the art and disclosed herein. Such an assay will generally comprisethe steps of: contacting test compounds and an IL-1 epsilon polypeptidewith an IL-1 epsilon counterstructure; determining whether the testcompounds affect the ability of IL-1 epsilon to bind thecounterstructure; and selecting one or more test compounds that affectthe ability ofIL-1 epsilon to bind the counterstructure. The inventivecombination methods further comprise evaluating selected compounds in asecond assay, for agonistic or antagonistic effect on biologicalactivity using one or more of the aforementioned assays.

Alternatively, the inventive combination methods may comprise a firstassay to determine whether a candidate molecule modulates a biologicalactivity of IL-1 epsilon, as described herein. According to suchcombination methods, molecules that modulate an IL-1 epsilon biologicalactivity in this manner are selected using one or more of theaforementioned assays for biological activity, and assayed to determinewhether the candidate molecule(s) bind IL-1 epsilon. The selectedmolecules may be tested to further define the exact region or regions ofIL-1 epsilon to which the test molecule binds (for example, epitopemapping for antibodies).

As disclosed previously, the types of assays for biological activitiesof IL-1 epsilon that can be used in the inventive combination methodsinclude assays for the expression of cytokines, assays for theexpression of cell-surface molecules, assays to detect activation ofsignaling molecules, assays to detect induction of mRNAs, and assaysthat evaluate cell proliferation or cell death (and combinationsthereof), as described herein. Molecules that bind and that have anagonistic or antagonistic effect on biologic activity will be useful intreating or preventing diseases or conditions with which thepolypeptide(s) are implicated.

Those of ordinary skill in the art understand that when the biologicalactivity observed in the presence of the test compound is greater thanthat observed when the test compound is absent, the test compound is anagonist of IL-1 epsilon, whereas when the biological activity observedin the presence of the test compound is less than that observed when thetest compound is absent, the test compound is an antagonist (orinhibitor) of IL-1 epsilon. Generally, an antagonist will decrease orinhibit, an activity by at least 30%; more preferably, antagonists willinhibit activity by at least 50%, most preferably by at least 90%.Similarly, an agonist will increase, or enhance, an activity by at least20%; more preferably, agonists will enhance activity by at least 30%,most preferably by at least 50%. Those of skill in the art will alsorecognize that agonists and/or antagonists with different levels ofagonism or antagonism respectively may be useful for differentapplications (i.e., for treatment of different disease states).

Homogeneous assays are mix-and-read style assays that are very amenableto robotic application, whereas heterogeneous assays require separationof free from bound analyte by more complex unit operations such asfiltration, centrifugation or washing. These assays are utilized todetect a wide variety of specific biomolecular interactions (includingprotein-protein, receptor-ligand, enzyme-substrate, and so on), and theinhibition thereof by small organic molecules. These assay methods andtechniques are well known in the art (see, e.g., High ThroughputScreening: The Discovery of Bioactive Substances, John P. Devlin (ed.),Marcel Dekker, New York, 1997 ISBN: 0-8247-0067-8). The screening assaysof the present invention are amenable to high throughput screening ofchemical libraries and are suitable for the identification of smallmolecule drug candidates, antibodies, peptides, and other antagonistsand/or agonists, natural or synthetic. Several useful assays aredisclosed in U.S. Ser. No. 09/851,673, filed May 8, 2001 (the relevantdisclosure of which is hereby incorporated by reference).

Candidate Molecules to be Tested for Modulation of IL-1 epsilonActivity:

The methods of the invention may be used to identify antagonists (alsoreferred to as inhibitors) and agonists of IL-1 epsilon activity fromcells, cell-free preparations, chemical libraries, cDNA libraries,recombinant antibody libraries (or libraries comprising subunits ofantibodies) and natural product mixtures. The antagonists and agonistsmay be natural or modified substrates, ligands, enzymes, receptors, etc.of the polypeptides of the instant invention, or may be structural orfunctional mimetics of IL-1 epsilon or its bindingpartner/counterstructure. Potential antagonists of the instant inventionmay include small molecules, peptides and antibodies that bind to andoccupy a binding site of the inventive polypeptides or a binding partnerthereof, causing them to be unavailable to bind to their natural bindingpartners and therefore preventing normal biological activity.Antagonists also include chemicals (including small molecules andpeptides) that interfere with the signaling pathways used by IL-1epsilon (for example, by inhibiting the interaction of receptorsubunits, or inhibiting the interaction of intracellular components ofthe signaling cascade). Potential agonists include small molecules,peptides and antibodies which bind to the instant polypeptides orbinding partners thereof, and elicit the same or enhanced biologiceffects as those caused by the binding of the polypeptides of theinstant invention. Moreover, substances that activate (or enhance) thesignaling pathways used by IL-1 epsilon are also included within thescope of agonists of IL-1 epsilon.

Small molecule agonists and antagonists are usually less than 10Kmolecular weight and may possess a number of physicochemical andpharmacological properties which enhance cell penetration, resistdegradation and prolong their physiological half-lives (Gibbs, J.,Pharmaceutical Research in Molecular Oncology, Cell, Vol. 79 (1994)).Antibodies, which include intact molecules as well as fragments such asFab and F(ab′)2 fragments, as well as recombinant molecules derivedtherefrom (including antibodies expressed on phage, intrabodies, singlechain antibodies such as scFv and other molecules derived fromimmunoglobulins that are known in the art), may be used to bind to andinhibit the polypeptides of the instant invention by blocking thepropagation of a signaling cascade. It is preferable that the antibodiesare humanized, and more preferable that the antibodies are human. Theantibodies of the present invention may be prepared by any of a varietyof well-known methods.

Additional examples of candidate molecules, also referred to herein as“test molecules” or “test compounds,” to be tested for the ability tomodulate IL-1 epsilon activity include, but are not limited to,carbohydrates, small molecules (usually organic molecules or peptides),proteins, and nucleic acid molecules (including oligonucleotidefragments typically consisting of from 8 to 30 nucleic acid residues).Peptides to be tested typically consist of from 5 to 25 amino acidresidues. Also, candidate nucleic acid molecules can be antisensenucleic acid sequences, and/or can possess ribozyme activity.

Small molecules to be screened using the hereindescribed screeningassays can typically be administered orally or by injection to a patientin need thereof. Small molecules that can be administered orally areespecially preferred. The small molecules of the invention preferablywill not be toxic (or only minimally toxic) at the doses required forthem to be effective as pharmaceutical agents, and they are preferablynot subject to rapid loss of activity in the body, such as the loss ofactivity that might result from rapid enzymatic or chemical degradation.In addition, pharmaceutically useful small molecules are preferably notimmunogenic.

The methods of the invention can be used to screen for antisensemolecules that inhibit the functional expression of one or more mRNAmolecules that encode one or more proteins that mediate an IL-1epsilon-dependent cellular response. An anti-sense nucleic acid moleculeis a DNA sequence that is capable of can hybridizing to the target mRNAmolecule through Watson-Crick base pairing, and inhibiting translationthereof. Alternatively, the DNA may be inverted relative to its normalorientation for transcription and so express an RNA transcript that iscomplementary to the target mRNA molecule (i.e., the RNA transcript ofthe anti-sense nucleic acid molecule can hybridize to the target mRNAmolecule through Watson-Crick base pairing). An anti-sense nucleic acidmolecule may be constructed in a number of different ways provided thatit is capable of interfering with the expression of a target protein.Typical anti-sense oligonucleotides to be screened preferably are 30-40nucleotides in length. The anti-sense nucleic acid molecule generallywill be substantially identical (although in antisense orientation) tothe target gene. The minimal identity will typically be greater thanabout 80%, but a higher identity might exert a more effective repressionof expression of the endogenous sequences. Substantially greateridentity of more than about 90% is preferred, though about 95% toabsolute identity would be most preferred.

Candidate nucleic acid molecules can possess ribozyme activity. Thus,the methods of the invention can be used to screen for ribozymemolecules that inhibit the functional expression of one or more mRNAmolecules that encode one or more proteins that mediate an IL-1 epsilondependent cellular response. Ribozymes are catalytic RNA molecules thatcan cleave nucleic acid molecules having a sequence that is completelyor partially homologous to the sequence of the ribozyme. It is possibleto design ribozyme transgenes that encode RNA ribozymes thatspecifically pair with a target RNA and cleave the phosphodiesterbackbone at a specific location, thereby functionally inactivating thetarget RNA. In carrying out this cleavage, the ribozyme is not itselfaltered, and is thus capable of recycling and cleaving other molecules.The inclusion of ribozyme sequences within antisense RNAs confersRNA-cleaving activity upon them, thereby increasing the activity of theantisense constructs.

The design and use of target RNA-specific ribozymes is described inHaseloff et al. (Nature, 334:585, 1988; see also U.S. Pat. No.5,646,023), both of which publications are incorporated herein byreference. Tabler et al. (Gene 108:175, 1991) have greatly simplifiedthe construction of catalytic RNAs by combining the advantages of theanti-sense RNA and the ribozyme technologies in a single construct.Smaller regions of homology are required for ribozyme catalysis,therefore this can promote the repression of different members of alarge gene family if the cleavage sites are conserved.

The following examples are presented to promote a fuller understandingof this invention. These examples do not, however, limit the scope ofthe invention.

EXAMPLE I Isolation and Identification of a New Human IL-1 Ligand

We screened a human genomic phage library (Stratagene catalog #946205)using a mixture of ³²P-labeled single-strand DNA probes corresponding tothe entire coding sequence of murine IL-1 epsilon. After low stringencywashing (low stringency washing is defined as 0.2× SSC/0.1% SDS, at roomtemperature, Ausubel et al. Current Protocols in Molecular Biology, Vol.2, p. 10.3, John Wiley & Sons, Inc., (1996)), a positive clone with astrong hybridization signal was identified. DNA made from this clone andsubjected to Southern analysis identified a 5.5 kb Sal I-Asp 718restriction fragment which was subcloned into pBluescript and sequenced.Homology analysis of the 5.5 kb fragment using the UWGCG computerprogram “bestfit” revealed that a 212 bp region within the clone was 74%similar at the nucleotide level to the 3 prime exon of murine IL-1epsilon. As set forth in FIG. 3, this 212 bp sequence contains an openreading frame of 70 amino acids with 66% similarity (64% identity) tothe 3 prime exon of murine IL-1 epsilon.

The genomic sequence around the human IL-1 epsilon locus was extendedanother 5 kb in the 5′ direction using a Genome Walking kit (availablefrom Clonetech) in accordance with manufacturer's instructions. Analysisof the sequence of this upstream region revealed three additionalputative exons. RT-PCR was used to confirm the expression of theseexons, and their linkage into a single IL-1 epsilon cDNA, in RNA fromfour different human tissue sources (thymus, tonsil, and the cell linesHL-60 and THP-1). Additionally, a cDNA clone was obtained from theStratagene Universal Human cDNA Library Array I that also demonstratedthe joining of the three 3′-most exons. The cDNA clone from theUniversal Human cDNA Library Array I was a partial clone that did notextend to the 5′ end of the open reading frame. Full-length human IL-1epsilon DNA sequences are disclosed in SEQ ID NO:7 and SEQ ID NO:12. Thepolypeptides encoded by SEQ ID NO:7 and SEQ ID NO:12 are disclosed inSEQ ID NO:8 and SEQ ID NO:13, respectively. As set forth in FIG. 4,amino acids 51-159 of SEQ ID NO:8 and SEQ ID NO:13 share 53% similarity(49% identity) with the murine IL-1 epsilon (long form).

EXAMPLE II Chromosome Mapping of Human IL-1 Epsilon by Radiation HybridMapping

PCR was performed using the Whitehead Institute/MIT Center for GenomeResearch Genebridge4 panel of 93 radiation the Whitehead Institute/MITCenter for Genome Research Genebridge4 panel of 93 radiation hybridswhich can be found by navigating to the Whitehead Institute/MIT website(www-genome.wi.mit, with an ‘edu’ extension), Searching the site forgenebridge4. Primers were used which lie within the putative 3 primeexon of human IL-1 epsilon and which amplify a 195 bp product from humangenomic DNA, but do not amplify hamster genomic DNA. The resuts of thePCRs were converted into a data vector that was submitted to theWhitehead/MIT Radiation Mapping site on the internet (www-seq.wi.mit,with an ‘edu’ extension). The data was scored and the chromosomalassignment and placement relative to known Sequence Tag Site (STS)markers on the radiation hybrid map was provide. According to theresults, human IL-1 epsilon lies on chromosome 2, at 11.54 cR from STSD2S121 and 4.3 cR from the marker CHLC.GAAT11C03. The WhiteheadInstitute/MIT web site provides additional information about radiationhybrid mapping.

EXAMPLE III Activation of Signaling Molecules in Human Cells by HumanIL-1 epsilon

The following describes tests and results that were carried out todetermine whether Il-1 epsilon is capable of activating some of the samesignaling molecules involved in stress responses as are activated byIL-1 alpha, IL-1 beta and other inflammatory cytokines.

Human IL-1 epsilon was transfected into COS-1 cells. Several days afterthe transfection, conditioned medium (containing the transientlyexpressed IL-1 epsilon) was harvested. Test cells were incubated withthis conditioned medium, or alternatively with conditioned medium fromCOS-1 cells transfected with the empty expression vector. Approximately10 minutes following the incubation, cell extracts were prepared fromthe test cells, and the presence of activated signaling molecules wasassayed by the use of antibodies specific for the phosphorylated formsof IKBalpha (phosphorylation on Ser32), p38 MAP kinase (phosphorylationon Thr180 and Tyr182), and Stress-Activated Protein Kinase (SAPK/JNK)(phosphorylation on Thr183/Tyr185) (the antibodies were obtained fromNew England Biolabs, Beverly, Mass.). These signal transductionmolecules are known to be involved in a wide range of cellular responsesto stimuli such as UV irradiation, endotoxin, and inflammatory cytokinesincluding IL-1 beta. Compared to control conditioned medium, conditionedmedium containing human Il-1 epsilon activated IKBalpha and p38 MAPkinase phosphorylation in a number of human cell lines including HumanForeskin Fibroblasts and Human Umbelical Vein Endothelial Cells (ATCCCRL-1730). In the non-Hodgkins lymphoma cell line K299, human IL-1epsilon specifically activated JNK/SAPK phosphorylation. These resultsdemonstrate that IL-1 epsilon is involved in stress response signalingpathways.

EXAMPLE IV Tissue Distribution of Human IL-1 Epsilon

The tissue distribution of human IL-1 epsilon MRNA was investigatedusing PCR amplification from a panel of first strand cDNA templates.Specifically, a Clontech (Palo Alto, Calif.) Human Multiple Tissue cDNAPanel was screened using a forward primer in exon 2 and a reverse primerin exon 4, which, together, amplify a 450 base-pair fragment of IL-1epsilon. The PCR reaction was run for 35 cycles with an annealingtemperature of 60 degrees C. PCR products were detected on an agarosegel using ethidium bromide.

Human IL-1 epsilon mRNA was detected in the spleen, lymph node, thymus,tonsil, and leukocyte tissues. The tissue with the highest levels ofhuman IL-1 epsilon mRNA is tonsil. Moreover, human IL-1 epsilon mRNA wasalso detected in small airway epithelium under certain conditions (inproliferative and Yersinia-infected, but not quiescent, cells), as wellas in the human cell lines MoT, HUT-102, Raji, THP-1, IMTLH, HL60, andHPT-4. Low levels of mRNA were detected in colon tissue as well as thecolon cell line T84, and in HUVEC treated with PMA and ionomycin. TheHaCAT keratinocyte cell line also expressed IL-1 epsilon mRNA aftertreatment with LPS, IL-1/TNF/IL-18, or ultraviolet light (at 30seconds).

Expression of IL-1 epsilon was also analyzed in several animal models ofhuman disease by conventional real-time polymerase chain reaction(RT-PCR) substantially as described in U.S. Ser. No. 09/876,790, filedJun. 6, 2001, and/or by TaqMan® RT-PCR (Applied Biosystems, Foster City,Calif.). Total RNA from small or large intestine (colitis models:DSS-induced colitis, anti-CD-3 induced ileitis and MdrKO spontaneouscolitis), spinal cord (multiple sclerosis [MS] models: EAE using SJLmice injected with PLP), or lung (asthma model: BALB/c/OVA-inducedasthma model) was used to make first strand cDNA. The level ofexpression was subjectively scored as a function of relative ethidiumbromide staining intensity.

Results of these experiments indicated that IL-1 epsilon was upregulatedin DSS-induced colitis in C57BL/6 mice, but not in BALB/c mice, or inanti-CD3 induced ileitis. Additionally, the expression of IL-1 epsilonwas upregulated in MdrKO mice that developed spontaneous colitis.Accordingly, IL-1 epsilon is implicated in the cause or prolongation ofinflammatory bowel disease, and antagonists thereof will be useful intreating or ameliorating inflammatory bowel disease in individualsafflicted with such conditions. Additionally, IL-1 epsilon was alsoupregulated in the OVA-induced asthma model, indicating that anantagonist thereof may be useful in treating or ameliorating asthma andother pulmonary conditions relating to an immune or inflammatoryresponse.

EXAMPLE V Activation of ICAM-1 Levels in Human Cells by Human IL-1Epsilon

The following describes tests and results that were carried out todetermine whether Il-1 epsilon is capable of activating some of the samecell surface molecules involved in stress responses as are activated byIL-1 alpha, IL-1 beta and other inflammatory cytokines.

Human IL-1 epsilon was transfected into COS-1 cells. Several days afterthe transfection, conditioned medium (containing the transientlyexpressed IL-1 epsilon) was harvested. Human foreskin fibroblast (HFF)cells were incubated for 18 hours at 37□C with this conditioned mediumdiluted 1:1 with fresh 0.5% serum-containing medium, or alternativelywith conditioned medium from control COS-1 cells transfected with theempty expression vector, diluted 1:1 with fresh 0.5% serum-containingmedium.

Following treatment with the conditioned medium from COS-1 cells, theHFF cells were washed twice with PBS and removed from the tissue culturevessel with versene (non-trypsin reagent). Cell-surface ICAM-1 levelswere measured by staining with anti-CD54-PE antibody (Pharmingen, SanDiego, Calif.) on ice for one hour followed by washing and FACS-baseddetection.

HFF cells incubated in conditioned medium from control COS-1 cellsexhibited a slight increase in ICAM-1 levels, relative to untreatedcells. On the other hand, HFF cells that were treated with conditionedmedium from COS-1 cells that had been transfected with epsilon exhibiteda two-fold increase in cell-surface ICAM-1 levels. The level of ICAM-1staining seen on the IL-1 epsilon treated HFF cells was comparable tothat induced on the same cells by purified IL-1 beta.

EXAMPLE VI Modulation of Cytokine Levels in Dendritic Cells by IL-1Epsilon

The following describes tests that were carried out to determine whetherIL-1 epsilon is capable of modulating cytokine secretion in dendriticcells.

Monocyte-derived dendritic cells (MODC) are obtained essentially asdescribed by Pickl et al. (J. Immunol. 157:3850, 1996). Briefly, highlypurified CD14(bright) peripheral blood monocytic cells are obtained fromperipheral blood using an AutoMACS cell sorting system and anti-CD14magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Themonocytic cells are cultured in the presence of IL-4 and GM-CSF forseven days to yield MoDC.

MoDC are treated for two to three days in the presence or absence ofIL-1 epsilon at varying concentrations; lipopolysaccharide (LPS) at10ng/ml is used as a positive control, and heat-inactivated IL-1 epsilon(heated at 100 degrees C. for 30 minutes) is used as a negative control.Cells are separated from the supernatant medium by centrifugation.

The supernatant medium is analyzed for soluble cytokine levels using asuitable assay (for example, the Luminex® multi-plex cytokine assay;Luminex Corporation, Austin, Tex.). Following two day culture, thesupernatant was harvested and assayed for several cytokines includingIL-10, IL-2, IL-4, IL-6, IL-8, IL-12 (p70 heterodimer), TNF-alpha,IFN-gamma, and GM-CSF.

The results of this assay indicated that IL-1 epsilon induced MoDC tosecrete significant levels of IL-10; secretion of IL-10was confirmed byELISA substantially as described below. The results indicated that IL-1epsilon-treated cells produced over 8 times more IL-10 as compared tocells treated with media only. This activity was observed in twoseparate experiments and was eliminated by heat denaturation (indicatingthat the increase in IL-10 was attributable to a heat-labile molecule,and not heat-stable LPS).

For analysis of the induction of cytokine mRNA, the cells are harvestedand total RNA is isolated (for example, using an RNeasy® Total RNASystem mini-kit, QIAGEN, Venlo, The Netherlands) and analyzed in asuitable, real-time quantitative polymerase chain reaction (PCR)analysis. Quantitative RT-PCR was performed using the ABI PRISM® 7700Sequence Detection System (Applied Biosystems, Foster City, Calif.) andTaqMan® reagents (Applied Biosystems). This analysis indicated that IL-1epsilon strongly induced MRNA levels of IL-1 alpha and IL-1 beta(approximately 10-15 fold above control). There was also a stronginduction of the p40 subunit of IL-12 but not the p35 subunit. A modestincrease in IL-10 and slight increases in Toll-like Receptors (TLR; Rocket al., Proc. Natl. Acad. Sci. USA 95:588, 1998; Hemmi et al., Nature408:740, 2000) 4,5, and 9, and B7-DC (Tseng et al., J. Exp. Med.193:839, 2001) was also observed. In all cases, heat inactivation ofIL-1 epsilon abolished the observed gene induction.

EXAMPLE VII Effect of IL-1 epsilon on Mixed Lymphocyte Reaction (MLR)

The following describes tests carried out to determine the effects ofIL-1 epsilon on TNF-alpha, IFN-gamma, and IL-1 secretion in a mixedleukocyte reaction (MLR) assay.

Briefly, MoDCs are generated as described above. Purified CD3+allogeneicT cells are obtained from freshly drawn blood using an AutoMACS cellsorting and anti-CD3 magnetic microbeads system (Miltenyi Biotec).

The allogeneic T cells are then mixed with MoDCs at a 1:10 MoDC:T ratioin quadruplicate in the presence of IL-1 epsilon at varyingconcentrations from 5 ng.ml to 200 ng/ml, or control preparations. Theensuing mixed lymphocyte reaction (MLR) is allowed to proceed for fourdays, and supernatants are harvested and assayed for TNF-alpha,IFN-gamma, and IL-10 using a suitable assay as described previously (forexample, the Luminex® multi-plex cytokine assay, DELFIA® or ELISAsubstantially as described below).

Using a DELFIA® to detect cytokines, it was found that IL-10 levels wereincreased above control using a dose of 5ng/ml IL-1 epsilon (untagged,produced in E. coli) and were further increased by addition of IL-1epsilon up to 200 ng/ml. At doses of 25 ng/ml and higher, IL-1 epsiloncaused increased TNF-alpha and interferon-gamma levels as well.

This activity was observed in two separate experiments and waseliminated by heat denaturation of the protein prep, indicating thatendotoxin contamination is not the cause. The positive control was amixture of 40 micrograms/ml SAC (heat killed Staphlyococcus aureus cells(Pansorbin), Calbiochem; La Jolla, Calif.) and 1 microgram/ml CD40L(Immunex Corporation, Seattle, Wash.), and the negative control wasmedia; a heat inactivated sample of IL-1 epsilon at 25 ng/ml was alsoincluded as a control for endotoxin contamination. A Flag®-polyHisversion of this protein did not read out in this assay when used at 25ng/ml.

EXAMPLE VIII Cytokine ELISA

The following describes an Enzyme-Linked Immunosorbent Assay (ELISA)that is useful to detect and/or quantitate secreted proteins. TheExample describes an assay specific for IL-10; those of skill in the artwill recognize that a similar assay cold be used to detect othermolecules.

ELISA plates (for example, Costar® EIA/RIA 96 well easy wash plates,Corning Incorporated Life Sciences, Acton, Mass.) were coated overnightwith 100 microliter of a 2 micrograms/ml mixture of Rat-anti-huIL-10capture antibody (BD Pharmingen, San Diego, Calif.) in binding solution(0.1M NaH₂PO₄, pH 9.0) at 4 degrees C. Plates were washed with washbuffer (phosphate buffered saline, or PBS, 0.5% Tween 20) four times(400 microliters/well/wash), then one time with PBS without Tween.Plates were blocked with 100 microliters of 5% non-fat dry milk in PBSfor 1 hour at room temperature (RT), and then washed with wash buffersix times.

Samples and controls were added to separate wells (100microliters/well); serial dilutions of a standard protein, recombinantHuIL-10 (BD Pharmingen) in PBS+3% BSA (starting at 10 ng/ml in 3-folddilutions through 7 points as a standard curve, with an eighth point asa blank) was used to generate a standard curve for quantitation. Theplates were incubated for one hour at RT, then washed with wash buffersix times as previously described, and incubated withbiotinylated-rat-anti-HuIL-10 (BD Pharmingen; 100 microliters/well of a200 ng/ml mixture in PBS+3% BSA) for one hour at RT. The plates werethen washed six times with wash buffer as before, andstreptavidin-conjugated horse radish peroxidase (SA-HRP; ZymedLaboratories, Inc., South San Francisco, Calif.; 100 microliters/well ofa 1:4000 dilution in PBS+3% BSA) was added.

After incubating at RT for 30 minutes, the plates were washed for thefinal time as described above, and color was developed by adding 100microliters/well of Tetramethylbenzidene (TMB) substrate (a 1:1 mixtureof TMB Peroxidase Substrate: Peroxidase Solution, Kirkegaard & PerryLaboratories, Inc., Gaithersburg, Md.). The plates were incubated for 30minutes at RT, at which time color development was stopped with 100microliters/well of 2N H₂SO₄. The plates were read at 450 nm wavelengthon a Molecular Dynamics (Molecular Dynamics, Sunnyvale, Calif.) platereader, a standard curve was prepared, and the quantity of IL-10 in thesamples determined by comparison to the standard curve.

EXAMPLE IX Cytokine DELFIA

The following describes a DELFIA® (dissociated enhanced lanthanidefluoroimmunoassay; PerkinElmer LifeSciences, Wallac Oy., Turku, Finland)that is useful to detect and/or quantitate secreted proteins. TheExample describes an assay specific for IL-10; those of skill in the artwill recognize that a similar assay could be used to detect othermolecules.

Briefly, DELFIA® plates (i.e., Costar® high binding 96-well plates,Coming Incorporated Life Sciences, Acton, Mass.) are coated with adetection (or capture) antibody (preferably a monoclonal antibody; 50microliters of antibody solution containing 2 micrograms antibody/ml inPBS) at 4 degrees C. for 24 hours. Plates are washed with wash buffer(phosphate buffered saline, or PBS, 0.05% Tween 20) four times (300microliters/well/wash), then used in an assay or stored.

Fifty microliters each of test supernatants and cell specific controlsare added to separate wells of an antibody-coated plate; dilutions ofstandard proteins are used to generate a standard curve forquantitation. Test supernatants and controls are incubated in theantibody coated plate to allow binding of cytokine to the antibody.Plates are then washed and a polyclonal biotinylated detection antibodyis added at a concentration of 10 nM in 50 microliters and incubated toallow binding to the captured cytokine. Plates are washed andStreptavidin-Europium (Eu) is added to the plate at a finalconcentration of 1 nM (0.06 micrograms/ml) in 50 microliters andincubated to allow binding to the biotinylated detection antibody.Plates are again washed and 100 microliters of enhancement solution isadded to bind the Eu. The Eu in solution is then detected by timeresolved fluorescence and the amount of cytokine secreted can bequantitated relative to standards which are added to each plate.

DELFIA® is amenable to full or partial automation (for example, using aSagian Bioassay core system, Beckman Coulter, Inc., Fullerton, Calif.,in combination with a plate reader such as a VICTOR2 TM, PerkinElmerLifeSciences), thereby rendering it useful for high-throughput testing.

EXAMPLE X Preparation of Antibodies to Human IL-1 Epsilon

Polyclonal antibodies are readily generated from a variety of sources,for example, horses, cows, goats, sheep, dogs, chickens, rabbits, mice,or rats, using procedures that are well-known in the art. In general,purified polypeptides of the invention, or a peptide based on the aminoacid sequence of polypeptides of the invention that is appropriatelyconjugated, is administered to the host animal typically throughparenteral injection. The immunogenicity of these polypeptides can beenhanced through the use of an adjuvant, for example, Freund's completeor incomplete adjuvant. Following booster immunizations, small samplesof serum are collected and tested for reactivity to the polypeptides.Examples of various assays useful for such determination include thosedescribed in: Antibodies: A Laboratory Manual, Harlow and Lane (eds.),Cold Spring Harbor Laboratory Press, 1988; as well as procedures such ascountercurrent immuno-electrophoresis (CIEP), radioimmunoassay,radio-immunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA),dot blot assays, and sandwich assays, see U.S. Pat. Nos. 4,376,110 and4,486,530.

Monoclonal antibodies are readily prepared using well-known procedures,see for example, the procedures described in U.S. Pat. Nos. RE 32,011,4,902,614, 4,543,439, and 4,411,993; Monoclonal Antibodies, Hybridomas:A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn,and Bechtol (eds.), 1980. Briefly, host animals, such as Balb/c mice,are injected intraperitoneally at least once, and preferably at leasttwice at about 3 week intervals with isolated and purified polypeptidesor conjugated polypeptides of the invention, optionally in the presenceof adjuvant. Preferably, at least about 10 μg of isolated and purifiedpolypeptide of the invention or peptides based on the amino acidsequence of polypeptides of the invention in the presence of RIBIadjuvant (RIBI Corp., Hamilton, Mont.) is used. Mouse sera are thenassayed by conventional dot blot technique or antibody capture (ABC) todetermine which animal produces the highest level of antibody and whosespleen cells are the best candidate for fusion.

Approximately two to three weeks later, the mice are given anintravenous boost of the polypeptides or conjugated polypeptides (suchas 3 μg suspended in sterile PBS). Mice are later sacrificed and spleencells fused with commercially available myeloma cells, such as Ag8.653(ATCC CRL-1580), following established protocols. Briefly, the myelomacells are washed several times in media and fused to mouse spleen cellsat a ratio of about three spleen cells to one myeloma cell. The fusingagent can be any suitable agent used in the art, for example,polyethylene glycol (PEG) or more preferably, 50% PEG: 10% DMSO (Sigma).The fusion is plated out into, for example, 96-well flat bottom plates(Corning) containing an appropriate medium, such as HAT supplementedDMEM media, and allowed to grow for about eight days. Supernatants fromresultant hybridomas are collected and added to, for example, a 96-wellplate for 60 minutes that is first coated with goat anti-mouse Ig.Following washes, ¹²⁵I-polypeptide or peptides of the invention areadded to each well, incubated for 60 minutes at room temperature, andwashed four times. Positive wells can be subsequently detected byconventional methods, such as autoradiography at −70 degrees C. usingKodak X-Omat S film. Other suitable means of identifying antibodies thatbind IL-1 epsilon may be used (including, for example, ELISA, IFA, orone of the aforementioned assays using cells that respond to IL-1epsilon).

Positive hybridoma cells can be injected intraperitoneally intosyngeneic rodents to produce ascites containing high concentrations (forexample, greater than 1 milligram per milliliter) of anti-IL-1 epsilonpolypeptides monoclonal antibodies. Alternatively, positive hybridomacells can be grown in bulk culture. Monoclonal antibodies aresubsequently purified, such as over a Protein A or G column (Pharmacia,Uppsala, Sweden) or by affinity chromatography.

Antibodies can be further tested to evaluate their effects on theability of IL-1 epsilon to induce a biological activity (for example,induction of inflammatory cytokines in MoDC, induction of ICAM-1 on HFFcells, phosphorylation of IKBalpha, p38 MAP kinase, and/orStress-Activated Protein Kinase (SAPK/JNK), or other markers of IL-1epsilon biological activity). An antibody that increases the ability ofIL-1 epsilon to induce a biological activity is referred to as anagonistic antibody, whereas an antibody that decreases the ability ofIL-1 epsilon to induce a biological activity is referred to as anantagonistic antibody. Both types of antibodies may be generated andidentified by means that are well known in the art, and will have usesin detection or purification of IL-1 epsilon, as reagents for researchor clinical use, and in therapy and/or diagnosis of conditions mediatedby IL-1 epsilon, as described herein.

EXAMPLE XI Mouse Inflammatory Bowel Disease Models

This example describes several mouse models of inflammatory boweldisease (IBD), which includes Crohn's Disease and ulcerative colitis.Inflammatory bowel disease in animals can either occur spontaneously orcan be experimentally induced. It is necessary to exercise care whenselecting IBD models to study to ensure that the particular modelselected appropriately represents the relevant stage of the inflammatoryprocess under investigation. Particularly useful models of IBD include:

A. Oral Administration of Dextran Sulfate Sodium (DSS)

The DSS induction model can be used to induce either chronic or acuteIBD. In the acute protocol, mice are given DSS (preferably with amolecular weight of 40 Kd; from 2% to 8%) in their drinking water forfrom one to eight days. The percent DSS and the duration of inductionwill vary depending on the strain of mouse used (for example,C3H/HeJ,C3H/HeJBir, NOD and NOD/SCID mice are highly susceptible, DBA/2,C57BL/6. BALB/c and 129/SvJ mice are moderately susceptible, withvarying degrees of susceptibility relative to each other, FVB mice aremoderately resistant, and NON/Ltj mice are resistant to DSS inducedcolitis). In the acute model, DSS is withdrawn after the inductionphase. To induce chronic colitis, 2-8% DSS is administered for from 5 toseven days followed by administration of water for ten days; this cycleis repeated three to four times.

DSS-induced colitis is marked by profound inflammation in the colon ofanimals characterized by crypt destruction, mucosal ulceration, erosionsand infiltration of lymphocytes and neutrophils into the mucosal tissue.Histopathologic changes are individually scored as 0 (no findings), 1(minimal), 2 (mild), 3 (moderate), 4 (severe) for each of the followingparameters: increased lymphocytes, increased neutrophils, ulceration,edema, crypt degeneration, and crypt regeneration. Total lesion score,crypt length and number of ulcers are also determined and used to gageseverity of colitis.

B. Anti-CD3-Induced Ileitis

Mice (for example, BALB/c, C57BL/6 or MPJ mice, 6-16 weeks of age) aregiven a single intraperitoneal (i.p.) injection of anti-CD3epsilonantibody or control Ab (50 micrograms diluted in 500 microliters PBS, pH7.4). In wildtype mice such as those listed above, this treatmentreliably induces diarrhea without being lethal. Immunosuppressants suchas cyclosporin A (CsA, 50 mg/kg) or dexamethasone (Dex, 50 mg/kg) may begiven i.p. either as a single dose at the same time as anti-CD3antibody, or daily for a total of three injections beginning at the timeof anti-CD3 injection, as control molecules that downregulate anyensuing immune response and prevent or ameliorate anti-CD3-inducedileitis.

Mice are monitored for clinical signs of ileitis; mice may be sacrificedat varying time points for histopathologic analysis and/or testing byother means to evaluate apoptosis in gut tissue. For histopathology,hematoxylin and eosin (H&E) stained tissue sections of paraffin embeddedintestinal specimens are graded in a blinded fashion, for example byusing a quantitative histology score based on the frequency of apoptoticepithelial cells within the epithelium and the ratio of villus height tocrypt length. Histological alterations of the small intestinal mucosathat may be observed include a reduced villus height, increasedthickness of the crypt region, loss of Paneth cells, goblet cells andIEL in the epithelial layer and severe morphologic changes of theepithelial cells. In the villi, the enterocytes may have lost theircolumnar and polarized morphology and become flattened. In the cryptregion, numerous apoptotic bodies may identified in the epithelium.

C. MdrKO Spontaneous Colitis

The MDR gene family was identified by an ability to confer multiple drugresistance in cell lines. Three genes have been identified in rodents(mdr1, mdr2 and mdr3), and two in humans (MDR1, MDR3). The mouse mdrlagene encodes a 170 kDa transmembrane protein that is expressed in manytissues, including intestinal epithelial cells and subsets of lymphoidand hematopoietic cells. Its function in these cells is currentlyunknown, however, mice deficient in mdr1a spontaneously develop colitis.In humans, MDR1 may be associated with IBD susceptibility (Satsangi etal., Nat. Genet. 14:199, 1996; Brant et al., Gastroenterology, 118:A331,2000), while decreased MDR1 expression has been reported in mucosaltissue from both CD and UC patients (Lawrance et al., Hum. Mol. Genet.10: 445, 2001; Farrell et al., Gastroenterology, 118:279, 2000). Mdr1aknockout mice (MdrKO) provide a model of both acute (spontaneous) andchronic (DSS-induced) IBD, similar to that seen in humans, where IBD isgenerally a mixture of both chronic and acute inflammation. Acutecolitis in MdrKO mice is marked by the spontaneous appearance ofdiarrhea and bloody stools in a subset of the mice; chronic colitis isinduced by administering 3% w/v DSS for seven days in drinking water,followed by normal water.

Histopathologic changes are individually scored as 0 (no findings), 1(minimal), 2 (mild), 3 (moderate), 4 (severe) for each of the followingparameters: increased mononuclear cells, increased neutrophils,ulceration, edema, crypt degeneration, and hyperplasia.

D. Helicobacter-Induced Colitis

Various strains of mice with immunologic defects (i.e., IL-10 ^(−/−)mice, recombinase-activating gene (Rag)1^(−/−) mice, T-cell receptoralpha (TCRalpha) ^(−/−) mice) are susceptible to colitis induced byinfection with Helicobacter spp., as described in Burich et al. (Am JPhysiol Gastrointest Liver Physiol 281:G764, 2001). Moreover, luminalbacteria appear to be an important factor contributing to thedevelopment of IBD in mice and humans. Accordingly, introduction ofHelicobacter spp. into immunodeficient mice also serves as an animalmodel of IBD humans (Burich et al. supra). In MdrKO mice, differentspecies of Helicobacter may have different effects on spontaneouscolitis; H. bilis infection induces IBD at a much earlier age, and thephenotypic appearance of Helicobacter-induced disease is similar, butnot identical, to spontaneous IBD. In contrast, there is minimal diseasein H. hepaticus-infected mdr1a−/− mice, and H. hepaticus appears todelay onset of spontaneous IBD. Accordingly, those of skill in the artcan utilize a Helicobacter-based model of IBD substantially as describedby Burich et al. supra.

EXAMPLE XII Mouse Asthma Models

This example describes a mouse model of asthma. Mice (for example,BALB/c) are sensitized with antigen (for example, ovalbumin [OVA]) byintraperitoneal injection of the antigen in alum. Several sensitizationschemes are known in the art; a preferred scheme is to inject 10micrograms of OVA three times at one week intervals (i.e., on day-21,day-14 and day-7). The mice are then challenged with antigen either byaerosol exposure (5% OVA) or intranasal administration (0.1 mg OVA). Thechallenge schedule may be selected from among shorter terms (i.e., dailychallenge on days 1, 2 and 3) or longer terms (i.e., weekly challengefor two to three weeks). The endpoints that are measured can includeairway hyperreactivity, bronchoalveolar lavage (BAL) cell number andcomposition, in vitro draining lung lymph node cytokine levels, serumIgE levels, and histopathologic evaluation of lung tissue. Other animalmodels of asthma are known, and include the use of other animals (forexample, C57BL/6 mice), sensitization schemes (for example, intranasalinoculation, use of other adjuvants or no adjuvants, etc.) and/orantigens (including peptides such as those derived from OVA or otherproteinaceous antigens, ragweed extracts or other extracts such as thoseused in desensitization regimens, etc.).

EXAMPLE XIII Mouse Collagen Induced Arthritis Model

This example describes two mouse models of rheumatoid arthritis, both ofwhich are induced by immunization with collagen (eg., collagen-inducedarthritis or CIA). One model is dependant on tumor necrosis factor(TNF), the other is TNF-independent. Those of skill in the art recognizethat other animals models of rheumatoid arthritis exist, and furtherthat various parameters within the models can be adjusted (see, forexample, Luross and Williams, Immunology 103:407, 2001; Schaller et al.,Nat Immunol 2:74, 2001; Bober et al., Arthritis Rheum 43:2660, 2000; orWeyand, C. M. in Rheumatology (Oxford) 2000 June, pgs:3-8)).

TNF-dependent CIA is induced in male, wild-type (wt) DBA/1 micesubstantially as a modification of the protocol described by Courtenay,.J. S. et al. (Nature 283:666, 1980) by immunization of mice with Type IIcollagen (CII; 100-200 micrograms) in complete Freund's adjuvant (CFA),followed by a booster of CII (200 micrograms) in incomplete Freund'sadjuvant (IFA) approximately three weeks later. In untreated mice, CIAmanifests in the paws, with increasing severity over time.

TNF-independent CIA is induced in male TNF Receptor double knockout(TNFR DKO) mice substantially as described above. TNFR DKO mice are micethat lack functional TNF receptors (both p55 and p75), and are describedin Peschon, et al. (J. Immunol. 160:943, 1998). Briefly, mice lackingfunctional p55 and p75 genes were generated in C57BL/6 background bygene targeting in embryonic stem cells. The TNFR DKO C57BL/6 mice wereback-crossed on to the DBA/1 genetic background to yield mice that werehomozygous for H-2q and were susceptible to development of CIA.

The severity of disease is judged by swelling and joint function of eachpaw, using a score from 0 to 4 (0=normal, no swelling; 1=swelling in 1to 3 digits; 2=mild swelling in ankles, forepaws or more than threedigits; 3=moderate swelling in multiple joints; 4=severe swelling withloss of function). The score for each paw is totaled for a cumulativescore for each mouse; cumulative scores are totaled for the mice in eachexperimental group to yield a mean clinical score.

EXAMPLE XIV Mouse Experimental Allergic Encephalomyelitis Model

This example describes two mouse models of demyelinating conditions;experimental autoimmune encephalomyelitis (or EAE) is designed toduplicate the secondary, immune mediated demyelination that occurs inmultiple sclerosis.

A. Myelin Oligodendrocyte Glycoprotein (MOG)-Induced EAE in C57BL/6 Mice

EAE is induced in female C57BL/6 mice substantially as described byMendel et al. (Eur. J. Immunol. 25:1951-59, 1995) by immunization ofmice with an antigen derived from rat myelin oligodendrocyteglycoprotein (preferably the MOG35-55 peptide described by Mendel etal., supra). Other encephalitogenic antigens may be used, including, forexample, whole spinal chord homogenate, purified whole myelin, myelinbasic protein, proteolipid protein, myelin associated glycoprotein,myelin-associated oligodendrocyte basic protein, or encephalitogenicpeptides derived from these antigens. The disease induction protocol ofMendel et al. may be modified to include the use of a lower dose ofMOG35-55 for immunization (see below), no booster immunization, and theuse of RIBI® adjuvant (Corixa Corporation, Seattle Wash.) instead ofcomplete Freund's adjuvant.

To induce EAE, groups of age and weight-matched mice are given a dose of100 micrograms of rat MOG35-55 emulsified in 0.2 ml RIBI® adjuvant andinjected subcutaneously (for example, at three sites distributed overthe shaved flank of a mouse). To induce EAE with accelerated onset, micemay be given an intravenous injection 500 ng pertussis toxin (ListBiological Laboratory Inc, Campbell, Calif.), administered 48 hoursafter administration of MOG35-55.

B. Proteolipid Protein (PLP)-Induced EAE in SJL Mice

The PLPISJL model results in a relapsing-remitting course of diseasethat mimics the course often seen in MS; however, SJL mice aresusceptible to anaphylaxis, and care must be given in choosing andadministering therapeutic agents to avoid induction of an anaphylacticresponse. EAE is induced in female SJL mice substantially as describedby McRae et al. et al. (J. Neuroimmunol. 38:229, 1992) by immunizationof mice with an antigen derived from rat proteolipid protein (preferablythe PLP13-151(S) peptide described by McRae et al., supra). Otherencephalitogenic antigens may be used, including, for example, wholespinal chord homogenate, purified whole myelin, myelin basic protein,proteolipid protein, myelin associated glycoprotein myelin-associatedoligodendrocyte basic protein, or encephalitogenic peptides derived fromthese antigens. The disease induction protocol of McRae et al. may bemodified as described above.

EAE is reliably induced in SJL/J mice actively immunized withPLP13-151(S) or another, suitable PLP-related antigen. Alternatively,EAE can be induced by adoptive transfer of PLP-specific T cells.

Administration of FIL1 antagonist(s) or control for either or bothmodels is initiated on the day after administration of theencephalitogenic peptide (day 1) and continued through day 11. Varyinginjection schedules can be used to evaluate the efficacy of the FIL1antagonist(s). Each mouse is injected intraperitoneally every other day(or according to the selected injection schedule) with 0.2 mlpyrogen-free phosphate-buffered saline (PBS) or 0.2 ml PBS containingFIL1 antagonist(s) or control. Endotoxin levels are monitored and mustbe less that <10 EU/mg of protein for all reagents. Mice are monitoreddaily for 30 to 35 days for weight loss, disease onset and severity ofclinical signs of EAE by an independent observer blinded to thetreatment groups.

The severity of EAE is assessed using either a standard EAE index systemin which “0” is used to indicate an asymptomatic mouse and clinicalscores ranging from 0.5 to 4 are used to indicate varying degrees ofascending paralysis, or a slightly modified version of the commonly usedEAE scoring system. In the latter system, “0” indicates a mouse with noevidence of disease and scores of 1-5 indicate varying degrees ofascending paralysis as follows: 1, tail paralysis; 2, hind limbweakness; 3, partial hind limb paralysis; 4, complete hind limbparalysis; 5, moribund or dead. The disease protocol described aboveinduces an acute episode of disease in control mice (peak score of 2-4)from which most recover at least partially. Thus the acute episode ofdisease is not lethal and mice do not reach a score of 5. Theaforedescribed scale may be modified to include a score of “0.5” whichis given to mice that show the earliest signs of EAE but that do notexhibit complete paralysis of the tail. Mice given a score of 0.5exhibit some or all of the following symptoms: overnight weight loss of1-2 grams; noticeable tremor when held up by the tail; and weakness atthe distal tip of the tail.

The median day of onset of EAE is determined by Kaplan-Meier Survivalanalysis. Significant differences in onset between groups are assessedusing a Log-Rank comparison. Fischer's exact test is used to analyze thestatistical significance of differences in the incidence of EAE amongthe groups of mice.

EXAMPLE XV Mouse Cuprizone-Induced Demyelinating Disease Model

This example describes a mouse model (cuprizone-induced demyelinatingdisease or CIDD) that is designed to mimic a type of demyelination thatoccurs in some cases of multiple sclerosis referred to as primarydemyelination. CIDD is induced by feeding cuprizone(bis-cyclohexanone-oxaldihydrazone, a copper chelator) to micesubstantially as described by Matsushima et al. (Brain Pathol. 11:107,2001). At low doses of cuprizone, mature oligodendrocytes in the CNS arespecifically insulted and they become unable to provide support formyelin. Demyelination occurs when the damaged myelin is stripped fromthe axons by microglia.

Some advantages of the CIDD model are that it reproducibly results inmassive demyelination in a large area of the mouse brain and it isreversible if cuprizone is removed from the diet. The model appears wellsuited for profiling gene expression during various stages ofdemyelination and remyelination. The model has been established inC57BL/6 mice, so it is also suitable for use in KO (knockout) or Tg(transgenic) mice with the B6 background. However, there are no obviousclinical signs associated with the demyelinating process, so analysismust be done by histology.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification, which arehereby incorporated by reference. The embodiments within thespecification provide an illustration of embodiments of the inventionand should not be construed to limit the scope of the invention. Theskilled artisan recognizes many other embodiments are encompassed by theclaimed invention.

1. A method for determining whether a test compound affects at least onebiological activity of an IL-1 epsilon polypeptide, the methodcomprising: a) contacting the test compound and the IL-1 epsilonpolypeptide with cells that exhibit the biological activity whencontacted with IL-1 epsilon; and, b) analyzing the cells for theoccurrence of the biological activity, wherein if the biologicalactivity observed in the presence of the test compound differs from thebiological activity that is observed when the test compound is absent,the test compound affects the biological activity of the IL-1 epsilon,wherein the biological activity is selected from the group consistingof: i. expression of one or more cytokines selected from the groupconsisting of IL-1 alpha, IL-1 beta, TNF-alpha, IL-10, IFN-gamma, IL-12p40, IL-6, ii. expression of one or more cell-surface molecules selectedfrom the group consisting of ICAM-1, TLR4, TLR5, TLR9, DC-B7; and iii.activation of one or more signaling molecules selected from the groupconsising of NFkappaB, p38 MAP kinase, Stress-Activated Protein kinase(SAPK/JNK), and further wherein the IL-1 epsilon polypeptide is selectedfrom the group consisting of: i′. a polypeptide comprising the aminoacid sequence of SEQ ID NO:8; ii′. a polypeptide comprising the aminoacid sequence of SEQ ID NO:13; iii′. a polypeptide that is at least 90%identical to SEQ ID NO:8, wherein the polypeptide activates IKBα or p38MAP kinase phosphorylation or the polypeptide activates cell surfaceexpression of ICAM-1; and iv′. a polypeptide that is at least 90%identical to SEQ ID NO:13, wherein the polypeptide activates IKBα or p38MAP kinase phosphorylation or the polypeptide activates cell surfaceexpression of ICAM-1.
 2. The method of claim 1, wherein the IL-1 epsilonpolypeptide is a fragment of the polypeptide of SEQ ID NO:8, wherein thefragment activates IKBα or p38 MAP kinase phosphorylation or thefragment activates cell surface expression of ICAM-1 and further whereinthe fragment has an amino terminus selected from the group consisting ofamino acids 1 through 5, and a carboxy terminus selected from the groupconsisting of amino acids 154 through 158, of SEQ ID NO:8.
 3. The methodof claim 1, wherein the IL-1 epsilon polypeptide is a fragment of thepolypeptide of SEQ ID NO:13, wherein the fragment activates IKBα or p38MAP kinase phosphorylation or the fragment activates cell surfaceexpression of ICAM-1 and further wherein the fragment has an aminoterminus selected from the group consisting of amino acids 1 through 5,and a carboxy terminus selected from the group consisting of amino acids154 through 158, of SEQ ID NO:13.
 4. The method of claim 1, wherein theIL-1 epsilon polypeptides are selected from the group consisting of SEQID NO:8 and SEQ ID NO:13.
 5. A method for testing a plurality of testcompounds to determine whether the test compounds affect at least onebiological activity of an IL-1 epsilon polypeptide, the methodcomprising: a) selecting test compounds that affect an ability of IL-1epsilon to bind an IL-1 epsilon counter structure; b) contacting theselected test compounds and an IL-1 epsilon polypeptide with cells thatexhibit a biological activity when contacted with IL-1 epsilon; and c)analyzing the cells for the occurrence of the biological activity,wherein if the biological activity observed in the presence of theselected test compound differs from the biological activity that isobserved when the selected test compound is absent, the selected testcompound affects the biological activity the IL-1 epsilon, wherein thebiological activity is selected from the group consisting of: iv.expression of one or more cytokines selected from the group consistingof IL-1 alpha, IL-1 beta, TNF-alpha, IL-10, IFN-gamma, IL-12 p40, 11-6,and combinations thereof; v. expression of one or more cell-surfacemolecules selected from the group consisting of ICAM-1, TLR4, TLR5,TLR9, DC-B7, and combinations thereof; and vi. activation of one or moresignaling molecules selected from the group consisting of NFkappaB, p38MAP kinase, Stress-Activated Protein Kinase (SAFK/JNK) and combinationsthereof, and further wherein the IL-1 epsilon polypeptide is selectedfrom the group consisting of: i′. a polypeptide comprising the aminoacid sequence of SEQ ID NO:8; ii′. a polypeptide comprising the aminoacid sequence of SEQ ID NO:13; iii′. a polypeptide that is at least 90%identical to SEQ ID NO: 8, wherein the polypeptide activates IKBα or p38MAP kinase phosphorylation or the polypeptide activates cell surfaceexpression of ICAM-1; and iv′. a polypeptide that is at least 90%identical to SEQ ID NO:13, wherein the polypeptide activates IKBα or p38MAP kinase phosphorylation or the polypeptide activates cell surfaceexpression of ICAM-1.
 6. The method of claim 5, wherein the IL-1 epsilonpolypeptide is a fragment of the polypeptide of SEQ ID NO:8, wherein thefragment activates IKBα or p38 MAP kinase phosphorylation or thefragment activates cell surface expression of ICAM-1 and further whereinthe fragment has an amino terminus elected from the group consisting ofamino acids 1 through 5, and a carboxy terminus selected from the groupconsisting of amino acids 154 through 158, of SEQ ID NO:8.
 7. The methodof claim 5, wherein the IL-1 epsilon polypeptide is a fragment of thepolypeptide of SEQ ID NO:13, wherein the fragment activity IKBα or p38MAP kinase phosphorylation or the fragment activates surface expressionof ICAM-1 and further wherein the fragment has an amino terminusselected from the group consisting of amino acids 1 through 5, and acarboxy terminus selected from the group consisting of amino acids 154through 158, of SEQ ID NO:13.
 8. The method of claim 5, wherein the IL-1epsilon polypeptides are selected from the group consisting of SEQ IDNO:8, and SEQ ID NO:13.